[11, 12] After transduction, luciferase/EGFP-expressing EGI-1 cells were transplanted by intraportal injection into 10 male SCID mice (6-8 weeks old; Charles River Laboratories Inc., Wilmington, MA). Further details are provided in the Supporting Materials. After the development of liver metastases, mice were sacrificed and tissue samples spanning the liver parenchyma were collected to perform IHC analyses to assess (1) the presence of tumor reactive stroma accompanying CCA liver implants, (2) involvement of EMT in the formation of tumor reactive stroma by EGFP coexpression and fluorescent in situ hybridization

(FISH) using both human and murine Y probes,[13] and (3) involvement of a PDGF-mediated cross-talk between CCA cells and CAFs. To study the functional Alvelestat price effects of PDGF in the cross-talk between cancer cells and fibroblasts, we assessed fibroblast proliferation (methyl tetrazolium salt [MTS] assay) and migration (Boyden chamber)

after direct stimulation with recombinant human (rh)PDGF-D (R&D Systems, Milan, Italy) at increasing doses (0.1, 1, 10, and 100 ng/mL), before and after administration of inhibitors of PDGFRβ, to determine a dose-response effect. PDGFRβ antagonism was achieved using the tyrosine kinase inhibitor, imatinib mesylate (1 µM for 24 hours; Cayman, Florence, Italy).[14] Furthermore, fibroblast Alectinib ic50 migration induced by rhPDGF-D (100 ng/mL) was evaluated after selective inhibition of the small Rho GTPases, RhoA, Rac1, and Cdc42, and of c-Jun N-terminal kinase (JNK) (see below). As specific inhibitors, we used NSC23766 (75 nM; Cayman) for Rac1,[15] CASIN (5 µM; Xcess Bioscience, San Diego, CA) for Cdc42,[16] Y-27632 (10 µM; Sigma-Aldrich, Milan, see more Italy) for RhoA/ROCK,[17] and SP600125 (10 µM; Sigma-Aldrich) for JNK.[18] Proliferation and migration of human fibroblasts were also evaluated after stimulation with conditioned media from CCA cells (EGI-1, TFK-1, and CCA1). Both experiments were run before and after addition of imatinib, and migration experiments

also after treatment with small interfering RNA (siRNA) for PDGF-D. siRNA for PDGF-D was performed in EGI-1 cells using RNAiMax and StealthsiRNA (Invitrogen, Milan, Italy). See the Supporting Materials for details. Human fibroblasts were exposed to increasing doses of rhPDGF-D (0.1, 1, 10, and 100 ng/mL) for 24 hours. Among the potential effectors of PDGF-D signaling, we assessed extracellular signal-regulated kinase 1/2 (ERK1/2) and JNK by western blotting of total cell lysates, and RhoA, Rac1, and Cdc42 by G-LISA. ERK1/2 (regulating cell proliferation) and the Rho GTPases (regulating cell migration) are downstream effectors of two major signaling pathways activated by PDGF-D, mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt), respectively.