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2007 Program and Abstracts | 2007 Posters
Pancreatic Cancer Pulls Down Lymphocyte Migration
Paola Fogar1, Daniela Basso4, Cristina Mazzon2, Eliana Greco3, Anna Valerio5, Tihana Kasic2, ELISA Fadi4, Filippo Navaglia4, Carlo-Federico Zambon1, Flaviano Favaro4, Stefania Schiavon4, Antonella Viola2, Mario Plebani3,4, Sergio Pedrazzoli*1
1Medical and Surgical Sciences, University of Padova, Padova, Italy; 2Biomedical Sciences, Venetian Institute of Molecular Medicine, Padova, Italy; 3Diagnostic Sciences and Special Therapies, University of Padova, Padova, Italy; 4Laboratory Medicine, University of Padova, Padova, Italy; 5Clinical and Experimental Medicine, University of Padova, Padova, Italy

In pancreatic cancer (PC) patients the immune response is not robust enough to counteract cell growth and metastases. Tumor cells may neutralize the immune system by evading detection and/or by inhibiting immune cells function. To test this latter hypothesis we verified in vitro whether PC cells conditioned media modify CD4+ T cell proliferation, migration and activation. CD4+ T lymphocytes were purified by negative selection using a RosetteSep kit (StemCell Technologies). CD4+ cells were cultured for 4 days in control medium or CAPAN1 (a PC cell line) fresh conditioned medium. Migration was assessed using a transwell system in the presence or absence of the chemoattractant hSDFα; the number of migrating cells was FACS counted. Proliferation was FACS analysed in control and conditioned CD4+ cells, labelled with carboxyfluorescein succinimidyl ester, after 72 hrs of co-culture with allogenic PBMC. Control and conditioned CD4+ cells were co-cultured for 24 hrs with unpulsed or staphylococcal enterotoxins (0.25, 0.5 and 1.0 μg/mL) pulsed EBV-B cells. In the supernatants interferon-γ (IFNγ) and IL4 were assayed. The number of control and conditioned migrating CD4+ T cells did not differ in the absence of hSDFα (t=1.0, p:ns). In the presence of hSDFα, migrating CAPAN1 conditioned lymphocytes were less (3337±390, mean±SEM) than control lymphocytes (6413±660) (t=7.55, p<0.001). CAPAN1 conditioned medium stimulated CD4+ T cell proliferation both in the presence (t=2.27, p<0.05) or absence (t=3.87, p<0.001) of allogenic PBMC. IL4 concentration did not significantly vary between conditioned or non conditioned APC stimulated CD4+ cells. Stimulation of CD4+ T cells by pulsed or unpulsed EBV-B induced higher IFNγ concentrations (pg/mL) in conditioned than in control cells (table 1). Control and conditioned media were ultrafiltered (mw cut-offs=30,000 and 10,000 Da). The effects of CAPAN1 conditioned medium on CD4+ T cell migration and IFNγ production were recovered in the mw fraction of >30.000 Da. In
Conclusion: we demonstrated that PC cells release soluble mediator/s of more than 30.000 Da which inhibit CD4+ T cell migration, activate proliferation and Th1 differentiation, supporting the hypothesis that tumor cells alter immune cells function.
Table 1

CD4+T + unpulsed EBV-B Mean±SEM CD4+T + 0.25ug/mL pulsed EBV-B Mean±SEM CD4+T + 0.5ug/mL pulsed EBV-B Mean±SEM CD4+T + 1.0ug/mL pulsed EBV-B Mean±SEM
Control 10.5±1.7 178.1±39.5 261.4±56.7 499.1±111.6
Conditioned 69.7±24.7 293.4±66.1 478.5±94.7 998.0±236.6
Student’st test for paired data t=2.56, p<0.05 t=2.92, p<0.05 t=3.30, p<0.05 t=2.82, p<0.05

2007 Program and Abstracts | 2007 Posters
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