Mesenchymal stromal cells priming with Autocrine Motility Factor increase their recruitment towards hepatocellular carcinoma

BAYO FINA JM; FIORE EJ; PICCIONI F; BOLONTRADE M; SGANGA L; MALVICINI M; PEIXOTO E; RIZZO M; ALANIZ L; AQUINO JB; ANDRIANI O; PODHAJCER O; GARCIA M; MAZZOLINI G

Vancouver

Congreso; 12th Annual Meeting - International Society for Stem Cell Research; 2014

ISSCR

Hepatocellular carcinoma (HCC) is the 3rd cause of cancer-related death worldwide. Unifortunately, its incidence and mortality are increasing steadily. Curative therapies can only be applied to a minority of patients. Particularly, several proinflammatory cytokines, chemokines and growth factors produced by tumor stroma have the ability to recruit the mesenchymal stromal cells (MSCs). However, the mechanisms involved in human MSC migration and anchorage to HCC are not fully elucidated. Moreover, MSCs were used as carriers of antitumoral genes towards HCC exploiting their capability to home into HCC; however enhancement of their recruitment into tumor is needed. Autocrine Motility Factor (AMF), a cytokine released by HCC cells, has been previously described to stimulate tumor cell motility, metalloproteinase (MMP) secretion, and enhancement of integrin â1 activity. Particularly, the AMF/AMFR pathway could be regulated by several proteins including Caveolin-1, Caveolin-2 and GDI-2. The aim of this study was analyze the role of AMF in MSC migration towards HCC. For that purpose, in vitro migration was studied by modified Boyden chamber, observing that MSCs not only migrated to recombinant AMF (rAMF) but also AMF blockage with an specific antibody reduced their migration toward conditioned medium (CM) derived from HCC tumors developed in nude mice (HuH7 y HC-PT-5). Similarly, zymography assays showed that MMP-2 activity was induced by the stimulation of MSCs with rAMF or HuH7-CM. Moreover, the specific blockage of AMF in HuH7-CM reduced the MMP-2 activity increase. Finally, MSCs were primed with rAMF (MSC-rAMF) and their migration capability was compared with unstimulated MSC. MSC-rAMF showed increased in vitro migration towards CM derived from both HCC tumors, and increased adhesion to endothelial cells. MSC-rAMF also had an induction in the mRNA levels of AMF receptor, MMP-3, caveolin-1, caveolin-2 and the inhibition of GDI-2. Western blot showed an increased level of AMFR, JNK, p-JNK, c-Fos, p-c-Fos and p-CREB in MSC-rAMF. In order to evaluate MSC in vivo migration, HCC cell line HuH7 was subcutaneously inoculated in nude mice and 10 days later CMDiI-DiR-labeled MSCs were intravenously injected. Three days later mice were sacrificed, tumor, liver, spleen and lung dissected and analyzed by Xenogen In Vivo Imaging System. In vivo migration showed that tumors from animals with MSC-rAMF have an increased signal in comparison with mice injected with unstimulated MSCs. Similar signal was observed in liver, lung and spleen from both groups. Moreover, the presence of CM-DiI (+) MSC in tumors was confirmed by fluorescent microscopy. Our results demonstrate that AMF plays a critical role in MSC recruitment to HCC, and its priming with rAMF enhances MSC migration to hepatocellular carcinoma, becoming a promising strategy to improve their therapeutic efficacy.