Several studies indicate that adult stem cells may improve the recovery from acute tissue injury. It has been suggested that they may contribute to tissue regeneration by the release of paracrine factors promoting proliferation of tissue resident cells. However, the factors involved remain unknown. In the present study we found that microvesicles (MVs) derived from human liver stem cells (HLSC) induced in vitro proliferation and apoptosis resistance of human and rat hepatocytes. These effects required internalization of MVs in the hepatocytes by an α4-integrin-dependent mechanism. However, MVs pre-treated with RNase, even if internalized, were unable to induce hepatocyte proliferation and apoptosis resistance, suggesting an RNA-dependent effect. Microarray analysis and quantitative RT-PCR demonstrated that MVs were shuttling a specific subset of cellular mRNA, such as mRNA associated in the control of transcription, translation, proliferation and apoptosis. When administered in vivo, MVs accelerated the morphological and functional recovery of liver in a model of 70% hepatectomy in rats. This effect was associated with increase in hepatocyte proliferation and was abolished by RNase pre-treatment of MVs. Using human AGO2, as a reporter gene present in MVs, we found the expression of human AGO2 mRNA and protein in the liver of hepatectomized rats treated with MVs. These data suggested a translation of the MV shuttled mRNA into hepatocytes of treated rats. In conclusion, these results suggest that MVs derived from HLSC may activate a proliferative program in remnant hepatocytes after hepatectomy by a horizontal transfer of specific mRNA subsets.

Invariant NKT (inv. NKT) cells co-express an invariant § g T cell receptor and the NK receptor NK1.1 and, upon CD1d-restricted recognition of the glycosphingolipid antigen § -galactosyl ceramide ( § GalCer), secrete large amounts of regulatory cytokines. We investigated whether § GalCer-dependent activation of inv. NKT cells protects from experimental autoimmune encephalomyelitis (EAE), an immune-mediated disease of the central nervous system mimicking multiple sclerosis, induced in C57BL/6 mice by the myelin oligodendrocyte glycoprotein (MOG) encephalitogenic peptide aa 35-55. § GalCer was administered at the time of immunization s.c., mixed with complete Freund's adjuvant and MOG35-55 peptide, or administered i.p., diluted in PBS. EAE onset was delayed and disease severity was decreased only when § GalCer was s.c. administered. The protective effect of s.c. administration of § GalCer was associated with a markedly enhanced IFN-+ production by liverconfined inv. NKT cells which, in turn, suppressed Th1-cytokine production and fostered secretion of IL-10 from MOG35-55-specific T cells. In vivo neutralization of IFN-+ , but not IL-4, reversed the protective effect induced by s.c. administration of § GalCer, further confirming the critical regulatory role exerted by IFN-+ -producing inv. NKT cells. Our results indicate that § GalCer, properly administered, may elicit an inv. NKT-cell-mediated suppressive effect on the effector function of encephalitogenic T cells; this effect is able to ameliorate autoimmune demyelination.

Cancer stem cells (CSCs) are responsible for tumor progression, metastases, resistance to therapy, and tumor recurrence. Therefore, the identification of molecules involved in CSC self-renewal is a necessary step toward more effective therapies. To this aim, through the transcription profiling of the murine ErbB2(+) tumor cell line TUBO vs. derived CSC-enriched mammospheres, Toll-like receptor 2 (TLR2) was identified as 2-fold overexpressed in CSCs, as confirmed by qPCR and cytofluorimetric analysis. TLR2 signaling inhibition impaired in vitro mammosphere generation in murine TUBO (60%) and 4T1 (30%) and human MDA-MB-231 (50%), HCC1806 (60%), and MCF7 (50%) cells. In CSC, TLR2 was activated by endogenous high-mobility-group box 1 (HMGB1), inducing IκBα phosphorylation, IL-6 and TGFβ secretion, and, consequently, STAT3 and Smad3 activation. In vivo TLR2 inhibition blocked TUBO tumor takes in 9/14 mice and induced a 2-fold reduction in lung metastases development by decreasing cell proliferation and vascularization and increasing apoptosis. Collectively, these results demonstrate that murine and human mammary CSCs express TLR2 and its ligand HMGB1; this autocrine loop plays a pivotal role in CSC self-renewal, tumorigenesis, and metastatic ability. These findings, while providing evidence against the controversial use of TLR2 agonists in antitumor therapy, lay out new paths toward the design of anticancer treatments.

Many preclinical studies of cancer immunotherapy are based on the testing of a single vaccination strategy in several tumor models. Moreover, most of those studies used xenogeneic Ags, which, owing to their high immunogenicity, may not represent realistic models for the validation of cancer immunotherapies. To address these issues, we compared the vaccination efficacy of three well established strategies (i.e., naked DNA; peptide-pulsed dendritic cells (DC), or a mixture of peptide and the Escherichia coli toxin LTR72) using the xenogeneic OVA or the naturally expressed tyrosinase-related protein 2 (TRP-2) tumor Ag in the B16 melanoma model. C57BL/6 mice received one to three s.c. injections of peptide-pulsed DC or DNA, or one to four mucosal administrations of peptide-toxin mixture. One to 2 wk later, the animals were challenged s.c. with B16 or B16 cells expressing OVA (B16-OVA). Vaccination of mice with OVA induced in all cases melanoma-specific CTL and protection against B16-OVA. When TRP-2 was used, all three vaccines elicited B16-specific CTL, but only DC pulsed with the immunodominant T cell epitope TRP-2181–188 allowed protection against B16. Even more importantly, a vaccination regimen with TRP-2-pulsed DC, started 24 h after the injection of a lethal number of B16 cells, caused a therapeutic effect in 60% of the challenged animals. Our results strongly emphasize the relevance of the tumor Ag in the definition of immunotherapeutic strategies for cancer, and support the use of peptide-pulsed DC as cancer vaccine in humans.

In an attempt to reveal deregulated miRNAs associated with the progression of carcinomas developed in BALB-neuT transgenic mice, we found increased expression of miR-135b during malignancy. Relevantly, we observed that miR-135b is up-regulated in basal or normal-like human breast cancers, and it correlates with patient survival and early metastatization. Therefore, we investigated its biological functions by modulating its expression (up- or down-regulation) in mammary tumor cells. Although no effect was observed on proliferation in cell culture and in orthotopically injected mice, miR-135b was able to control cancer cell stemness in a mammosphere assay, anchorage-independent growth in vitro, and lung cancer cell dissemination in mice after tail vein injections. Focusing on the miR-135b molecular mechanism, we observed that miR-135b controls malignancy via its direct targets, midline 1 (MID1) and mitochondrial carrier homolog 2 (MTCH2), as proved by biochemical and functional rescuing/phenocopying experiments. Consistently, an anti-correlation between miR-135b and MID1 or MTCH2 was found in human primary tumor samples. In conclusion, our research led us to the identification of miR-135b and its targets, MID1 and MTCH2, as relevant coordinators of mammary gland tumor progression.