DataSheet_1_Arachidonate 15-lipoxygenase-mediated production of Resolvin D5n-3 DPA abrogates pancreatic stellate cell-induced cancer cell invasion.doc

Activation of pancreatic stellate cells (PSCs) to cancer-associated fibroblasts (CAFs) is responsible for the extensive desmoplastic reaction observed in PDAC stroma: a key driver of pancreatic ductal adenocarcinoma (PDAC) chemoresistance leading to poor prognosis. Specialized pro-resolving mediators (SPMs) are prime modulators of inflammation and its resolution, traditionally thought to be produced by immune cells. Using liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based lipid mediator profiling PSCs as well as primary human CAFs express enzymes and receptors to produce and respond to SPMs. Human PSC/CAF SPM secretion profile can be modulated by rendering these cells activated [transforming growth factor beta (TGF-β)] or quiescent [all-trans retinoic acid (ATRA)]. ATRA-induced nuclear translocation of arachidonate-15-lipoxygenase (ALOX15) was linked to increased production of n-3 docosapentaenoic acid-derived Resolvin D5 (RvD5n-3 DPA), among other SPMs. Inhibition of RvD5n-3 DPA formation increases cancer cell invasion, whereas addback of this molecule reduced activated PSC-mediated cancer cell invasion. We also observed that circulating concentrations of RvD5n-3 DPA levels were decreased in peripheral blood of metastatic PDAC patients when compared with those measured in plasma of non-metastatic PDAC patients. Together, these findings indicate that RvD5n-3 DPA may regulate cancer–stroma cross-talk and invasion.</p

DataSheet_2_Arachidonate 15-lipoxygenase-mediated production of Resolvin D5n-3 DPA abrogates pancreatic stellate cell-induced cancer cell invasion.pdf

Activation of pancreatic stellate cells (PSCs) to cancer-associated fibroblasts (CAFs) is responsible for the extensive desmoplastic reaction observed in PDAC stroma: a key driver of pancreatic ductal adenocarcinoma (PDAC) chemoresistance leading to poor prognosis. Specialized pro-resolving mediators (SPMs) are prime modulators of inflammation and its resolution, traditionally thought to be produced by immune cells. Using liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based lipid mediator profiling PSCs as well as primary human CAFs express enzymes and receptors to produce and respond to SPMs. Human PSC/CAF SPM secretion profile can be modulated by rendering these cells activated [transforming growth factor beta (TGF-β)] or quiescent [all-trans retinoic acid (ATRA)]. ATRA-induced nuclear translocation of arachidonate-15-lipoxygenase (ALOX15) was linked to increased production of n-3 docosapentaenoic acid-derived Resolvin D5 (RvD5n-3 DPA), among other SPMs. Inhibition of RvD5n-3 DPA formation increases cancer cell invasion, whereas addback of this molecule reduced activated PSC-mediated cancer cell invasion. We also observed that circulating concentrations of RvD5n-3 DPA levels were decreased in peripheral blood of metastatic PDAC patients when compared with those measured in plasma of non-metastatic PDAC patients. Together, these findings indicate that RvD5n-3 DPA may regulate cancer–stroma cross-talk and invasion.</p

Altered expression of genes encoding proteins closely related to IGF1 physiology.

<p>Altered expression of genes encoding proteins closely related to IGF1 physiology.</p

qPCR-RT measurement of RNA (cDNA) expression of genes encoding proteins closely related to IGF-1 function.

<p>*p<0.05, ** p 0.01 vs. controls (Wt group). <b>(A)</b> <i>Igf1;</i> <b>(B), (C), (D), (E), (F)</b> <i>Igfbp3</i>, <i>4</i>, <i>5</i>, and, 7, respectively; <b>(G)</b> <i>Ctgf/Igfbp8</i>. n = 10, each group.</p

Hemodynamic values in perfused hearts from the three experimental groups.

<p>dP/dt (mmHg/s), expressing left ventricular contractility, before and after I/R. Before I/R IGF-1 deficient mice showed a reduction of dP/dt but did not reach statistical significance, whereas Hz+IGF-1 mice presented quite similar values to controls (Wt mice). After, I/R, hearts from controls showed a significant reduction in contractility (dP/dt) and similar results were observed in hearts from Hz+IGF-1 group. However, no response after I/R was found in untreated IGF-1 deficient mice (p = ns). ## p<0.01 controls after I/R vs before I/R; # p<0.05 Hz+IGF-1 after I/R vs the same group before I/R (n = 5 each group).</p

qPCR-RT measurement of RNA (cDNA) expression for coded proteins involved in heart structure, function, and pathology.

<p><b>(C), (E), (F),</b> and <b>(G)</b> show low expression in both groups of IGF-1 deficient mice for <i>Myl7</i>, <i>S100a8</i>, <i>S100a9</i>, <i>Nppa</i>, respectively; <b>(H), (I), (J),</b> and <b>(L)</b> depict a reduced expression among the untreated Hz group and some recovering with the substitutive treatment (Hz+IGF-1) for <i>Nppb</i>, <i>Npr3</i>, <i>Snca</i>, <i>Acta2</i>, respectively; <b>(K)</b> shows a marked genetic overexpression for <i>Osbpl6</i> in both Hz group of animals. *p<0.05, ** p 0.01 vs. controls (Wt group); & p<0.05 Hz+IGF-1 group vs untreated Hz mice (n = 10 per group).</p

Hemodynamic values in perfused hearts from controls, mice with IGF-1 deficiency (Hz) and Hz treated with IGF-1 therapy.

<p>Hemodynamic values in perfused hearts from controls, mice with IGF-1 deficiency (Hz) and Hz treated with IGF-1 therapy.</p

Expression of genes encoding proteins involved on inflammation, extracellular matrix regulation, and in heart metabolism.

<p>Expression of genes encoding proteins involved on inflammation, extracellular matrix regulation, and in heart metabolism.</p

Coronary vasoconstriction to Ang II.

<p><b>(A)</b> Coronary perfusion pressure (mmHg) in hearts from the three experimental groups, at different concentrations (10⁻¹⁰ to 10⁻⁷) of Ang II before Ischemia/Reperfusion: Ang II induced vasoconstriction dose-dependent on perfused hearts from controls and Hz+IGF-1, increasing coronary perfusion pressure. However, no effect was observed after Ang II injection in hearts from Hz group. <b>(B)</b> Coronary perfusion pressure after I/R: vasoconstriction to Ang II was reduced both in controls and Hz+IGF-1 group. But no response was observed in untreated Hz group, showing a quite total insensibility to AngII (n = 5 per group). * p<0.05 Hz vs Controls; £ p<0.05 after vs. before I/R in the same group (control or Hz+IGF-1).</p

qPCR-RT measurement of RNA (cDNA) expression for coded proteins involved in inflammation, extracellular matrix regulation, and heart metabolism.

<p><b>(B), (C), (F),</b> and <b>(I)</b> represent those genes (<i>Timp2</i>, <i>Timp3</i>, <i>Fn1</i>, <i>Pdk1</i>, respectively) whose expression is downregulated by IGF-1 deficiency and recovered by substitutive treatment; <b>(A), (G),</b> and <b>(H)</b> refer to those genes (<i>C3</i>, <i>Aqp4</i>, <i>Ahsg</i>, respectively) underexpressed in both Hz groups; <b>(D)</b> shows that <i>Serpine1</i> was overexpressed in Hz animals and contrarily underexpressed when substitutive treatment was applied; and <b>(E)</b> illustrates how <i>Serpinh1</i> is upregulated by IGF-1 deficiency and how IGF-1 substitutive therapy normalizes its expression (n = 10 animals per group). *p<0.05, ** p 0.01 vs. controls (Wt group); & p<0.05 Hz+IGF-1 group vs. untreated Hz mice.</p