Elisa Fadi

Pancreatic cancer biomarkers discovery by surface-enhanced laser desorption and ionization time-of-flight mass spectrometry.

Abstract Background: Surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF/MS), a laboratory-friendly technique, is used to identify biomarkers for cancer. The aim of the present study was to explore the application of SELDI proteomic patterns in serum for distinguishing between cases of pancreatic cancer, chronic pancreatitis, type 2 diabetes mellitus and healthy controls. Methods: Sera from 12 healthy controls, 24 patients with type 2 diabetes mellitus, 126 with pancreatic cancer, including 84 with diabetes, and 61 with chronic pancreatitis, 32 of which were diabetics, were analyzed using SELDI-TOF/MS. Spectra (IMAC-30) were clustered and classified using Biomarker Wizard and Biomarker Pattern software. Results: Two decision tree classification algorithms, one with and one without CA 19-9, were constructed. In the absence of CA 19-9, the splitting protein peaks were: m/z 1526, 1211, and 3519; when CA 19-9 was used in the analysis, it replaced the m/z 3519 splitter. The two algorithms performed equally for classifying patients. A classification tree that considered diabetic patients only was constructed; the main splitters were: 1211, CA 19-9, 7903, 3359, 1802. With this algorithm, 100% of patients with type 2 diabetes mellitus, 97% with chronic pancreatitis and 77% of patients with pancreatic cancer were correctly classified. SELDI-TOF/MS features improved the diagnostic accuracy of CA 19-9 (AUC=0.883 for CA 19-9; AUC=0.935 for CA 19-9 and SELDI-TOF/MS features combined). Conclusions: SELDI-TOF/MS allows identification of new peptides which, in addition to CA 19-9, enable the correct classification of the vast majority of patients with pancreatic cancer, which can be distinguished from patients with chronic pancreatitis or type 2 diabetes mellitus. Clin Chem Lab Med 2009;47:713–23.

Pancreatic Tumors and Immature Immunosuppressive Myeloid Cells in Blood and Spleen: Role of Inhibitory Co-Stimulatory Molecules PDL1 and CTLA4. An In Vivo and In Vitro Study

Background Blood and spleen expansion of immature myeloid cells (IMCs) might compromise the immune response to cancer. We studied in vivo circulating and splenic T lymphocyte and IMC subsets in patients with benign and malignant pancreatic diseases. We ascertained in vitro whether pancreatic adenocarcinoma (PDAC)-associated IMC subsets are induced by tumor-derived soluble factors and whether they are immunosuppressive focusing on the inhibitory co-stimulatory molecules PDL1 and CTLA4. Methodology and Principal Findings 103 pancreatic and/or splenic surgical patients were enrolled including 52 PDAC, 10 borderline and 10 neuroendocrine tumors (NETs). Lymphocytes and IMCs were analysed by flow cytometry in blood, in spleen and in three PDAC cell conditioned (CM) or non conditioned PBMC. PDL1 and CTLA4 were studied in 30 splenic samples, in control and conditioned PBMC. IMCs were FACS sorted and co-coltured with allogenic T lymphocytes. In PDAC a reduction was found in circulating CD8+ lymphocytes (p = 0.004) and dendritic cells (p = 0.01), which were reduced in vitro by one PDAC CM (Capan1; p = 0.03). Blood myeloid derived suppressive cells (MDSCs) CD33+CD14−HLA-DR− were increased in PDAC (p = 0.022) and were induced in vitro by BxPC3 CM. Splenic dendritic cells had a higher PDL1 expression (p = 0.007), while CD33+CD14+HLA-DR− IMCs had a lower CTLA4 expression (p = 0.029) in PDAC patients. In vitro S100A8/A9 complex, one of the possible inflammatory mediators of immune suppression in PDAC, induced PDL1 (p = 0.018) and reduced CTLA4 expression (p = 0.028) among IMCs. IMCs not expressing CTLA4 were demonstrated to be immune suppressive. Conclusion In PDAC circulating dendritic and cytotoxic T cells are reduced, while MDSCs are increased and this might favour tumoral growth and progression. The reduced CTLA4 expression found among splenic IMCs of PDAC patients was demonstrated to characterize an immune suppressive phenotype and to be consequent to the direct exposure of myeloid cells to pancreatic cancer derived products, S100A8/A9 complex in particular.

Suicide gene therapy with the yeast fusion gene cytosine deaminase/uracil phosphoribosyltransferase is not enough for pancreatic cancer.

Objectives: Suicide gene therapy with FCY1 gene, encoding cytosine deaminase (CD), together with FUR1, encoding uracil phosphoribosyltransferase (UPRT), has been proposed for pancreatic cancer therapy in vivo. We ascertained whether gene therapy with FCY1-FUR1 is effective in killing pancreatic cancer cells after 5-fluorocytosine (5-FC) treatment. Methods: AsPC1, BxPC3, Capan1, MIA PaCa2, and Panc1 cell lines were transfected using 2 plasmid vectors expressing CD only (pRSV-CD) or the chimera CD-UPRT (pRSV-CD-UPRT). Control and pRSV-CD- or pRSV-CD-UPRT-transfected cell lines were treated with 0, 0.1, 0.5, 1, 5, and 10 mM of 5-FC for 1, 3, 6, 8, 10, and 13 days. Results: FCY1 alone did not confer sensitivity to 5-FC. The CD-UPRT-transfected BxPC3 and Panc1 were sensitive to very low 5-FC doses (0.1 mM). 5-Fluorocytosine-sensitive transfected cell lines rapidly converted 5-FC into 5-fluorouracil, whereas the 5-FC resistant cell lines had an impaired 5-FC conversion. Conclusions: Suicide gene therapy with the FCY1 gene alone was ineffective in the treatment of pancreatic cancer in vitro. The pRSV-CD-UPRT construct conferred 5-FC sensitivity to some pancreatic cancer cell lines. Therefore, the application in vivo of suicide gene therapy with FCY1 alone or in combination with the FUR1 gene is probably destined to fail.

Pancreatic cancer alters human CD4+ T lymphocyte function: a piece in the immune evasion puzzle.

Objectives: To verify whether the dysregulation of CD4+ T cells concurs in worsening the outcome of pancreatic cancer, we compared the effects of pancreatic cancer and other gastrointestinal cancer cell-conditioned media on the (1) proliferation, migration, and differentiation of CD4+ T cells and (2) expansion of CD4+ memory (CD45RO), naive (CD45RA), activated (CD69), and regulatory (CD25) subsets. Methods: After culture of CD4+ T cells in control, pancreatic (BxPC3, Capan1, MiaPaCa2), or gastrointestinal cancer (AGS, HepG2, HT29) cell-conditioned media, we evaluated proliferation, migration, interferon &ggr; (IFN&ggr;) production, and CD45RA, CD45RO, CD69, and CD25 membrane expression in control and conditioned CD4+ T cells. Results: Only pancreatic cancer-conditioned media (1) inhibited CD4+ T-cell proliferation (P < 0.001) and migration under human stromal cell-derived factor-&agr; chemotaxis (P < 0.001) and (2) induced CD4+ T-cell IFN&ggr; production (P < 0.05) and the expansion of the CD69-positive subset (P < 0.001) with respect to the control, with no changes being found in the CD45RA, CD45RO, and CD25 subsets. Conclusions: The in vitro findings achieved in the present study demonstrate that pancreatic cancer cells inhibit CD4+ T-cell proliferation and migration, induce IFN&ggr; production, and favor a CD69+ subset expansion, suggesting that CD4+ T cells play an important role in pancreatic cancer immune evasion.

Altered intracellular calcium fluxes in pancreatic cancer induced diabetes mellitus: Relevance of the S100A8 N‐terminal peptide (NT‐S100A8)

After isolating NT‐S100A8 from pancreatic cancer (PC) tissue of diabetic patients, we verified whether this peptide alters PC cell growth and invasion and/or insulin release and [Ca2+]i oscillations of insulin secreting cells and/or insulin signaling. BxPC3, Capan1, MiaPaCa2, Panc1 (PC cell lines) cell growth, and invasion were assessed in the absence or presence of 50, 200, and 500 nM NT‐S100A8. In NT‐S100A8 stimulated β‐TC6 (insulinoma cell line) culture medium, insulin and [Ca2+] were measured at 2, 3, 5, 10, 15, 30, and 60 min, and [Ca2+]i oscillations were monitored (epifluorescence) for 3 min. Five hundred nanomolars NT‐S100A8 stimulated BxPC3 cell growth only and dose dependently reduced MiaPaCa2 and Panc1 invasion. Five hundred nanomolars NT‐S100A8 induced a rapid insulin release and enhanced β‐TC6 [Ca2+]i oscillations after both one (F = 6.05, P < 0.01) and 2 min (F = 7.42, P < 0.01). In the presence of NT‐S100A8, [Ca2+] in β‐TC6 culture medium significantly decreased with respect to control cells (F = 6.3, P < 0.01). NT‐S100A8 did not counteract insulin induced phosphorylation of the insulin receptor, Akt and IκB‐α, but it independently activated Akt and NF‐κB signaling in PC cells. In conclusion, NT‐S100A8 exerts a mild effect on PC cell growth, while it reduces PC cell invasion, possibly by Akt and NF‐κB signaling, NT‐S100A8 enhances [Ca2+]i oscillations and insulin release, probably by inducing Ca2+ influx from the extracellular space, but it does not interfere with insulin signaling. J. Cell. Physiol. 226: 456–468, 2011.

Analogs of vitamin E epitomized by alpha-tocopheryl succinate for pancreatic cancer treatment: in vitro results induce caution for in vivo applications.

Objectives: &agr;-Tocopheryl succinate (&agr;-TOS) is thought to be toxic only for cancer cells. We ascertained in vitro &agr;-TOS effects on pancreatic cancer (PC) and normal cell growth and verified whether the combination of nontoxic &agr;-TOS and 5-fluorouracil (5-FU) doses causes cancer cell death and whether &agr;-TOS effects are mediated by the proapoptotic proteins Bax/Bak and/or SMAD4/DPC4 status. Methods: Five PC cell lines, myoblasts, normal monocytes, wild-type (WT) and Bax/Bak double knockout mouse embryonic fibroblast (MEF) cells, and permanently SMAD4/DPC4-transfected PSN1 cells were cultured in 1% and 10% fetal calf serums (FCSs), without or with &agr;-TOS (5-500 &mgr;mol/L). Nontoxic 5-FU (0.0001 mmol/L) and &agr;-TOS alone or in combination were also evaluated. Results: Only PSN1 PC cell line, which had SMAD4/DPC4 homozygous deletion, was sensitive to nontoxic &agr;-TOS doses (5 &mgr;mol/L in 1% FCS and 50 &mgr;mol/L in 10% FCS). A 20-&mgr;mol/L &agr;-TOS inhibited MEF-WT, not MEF-double knockout growth. Only PSN1 cells were sensitive to nontoxic 5-FU and &agr;-TOS combination. SMAD4/DPC4 transfection restored PSN1 resistance to the effects of combined 5-FU and &agr;-TOS effects. Conclusions: Only a minority of PC cells are sensitive to the antiproliferative effects of &agr;-TOS, any sensitivity appearing to be correlated with SMAD4/DPC4 homozygous deletion and Bax/Bak expression.