Autofluorescence properties of murine embryonic stem cells during spontaneous differentiation phases.

BACKGROUND AND OBJECTIVE: The autofluorescence (AF) analysis allows in vivo, real-time assessment of cell functional activities, depending on the presence of biomolecules strictly involved in metabolic reactions and acting as endogenous fluorophores. Pluripotent stem cells during differentiation are known to undergo changes in their morphofunctional properties, with particular reference to bioenergetic metabolic signatures involving endogenous fluorophores such as NAD(P)H, flavins, lipofuscin-like lipopigments. Since the development of regenerative therapies based on pluripotent cells requires a careful monitoring of the successful maturation into the desired phenotype, aim of our work is to evaluate the AF potential to assess the differentiation phases in a murine stem cell model. STUDY DESIGN/MATERIALS AND METHODS: Mouse embryonic stem cells (ESCs) maintained with and without leukemia inhibitory factor (LIF), embryoid bodies (EBs), and EB-derived cells undergoing spontaneous differentiation toward the hematopoietic lineage have been used as a sample models. Cell AF properties have been characterized upon 366-nm excitation, under living conditions and in the absence of exogenous markers. Imaging, microspectrofluorometric techniques, and spectral fitting analysis based on the spectral parameters of each endogenous fluorophore have been applied to estimate their contribution to the whole cell AF emission spectra. Specific cytochemical labeling has been performed to validate AF data. RESULTS: Depending on the differentiation phases, cells undergo changes in morphology, AF distribution patterns, and AF emission spectral profiles. These latter reflect variations in the single endogenous fluorophore contribution to the overall emission. The coenzyme NAD(P)H accounts for up to 80% of the whole spectral area. The free form prevails on the bound one, and their changes have been investigated in terms of NAD(P)Hbound/free and redox ratios. These values vary in agreement with a slow metabolic activity and prevailing glycolytic metabolism in the undifferentiated HM1 cells, an increased metabolic activity still relying on glycolysis during the early differentiation phases, and an increased oxidative phosphorylation in EB and hematopoietic precursor cells. Lipofuscin-like lipopigments decrease following differentiation, and porphyrins contributing for less than 5%, prevail in the more actively differentiating cells. These results reflect the shift between anaerobic and aerobic respiration following differentiation, consistently with a decreased autophagy of cell organelles (i.e., mitochondria, as a strategy reported in the literature to keep the undifferentiated homeostasis state), higher mitochondrial activity with more numerous NADH binding sites and synthesis of heme as prosthetic group of proteins, that is, cytochromes. CONCLUSIONS: These data open promising perspectives for the monitoring of stem cells differentiation under living conditions without labeling with exogenous agents (inducing perturbations when used in vivo), or immunomarkers not always available for veterinary and zootechnics, by exploiting endogenous fluorophores as intrinsic biomarkers of cell morphofunctional changes.

Monovalency unleashes the full therapeutic potential of the DN-30 anti-Met antibody.

Met, the high affinity receptor for hepatocyte growth factor, is one of the most frequently activated tyrosine kinases in human cancer and a validated target for cancer therapy. We previously developed a mouse monoclonal antibody directed against the extracellular portion of Met (DN-30) that induces Met proteolytic cleavage (receptor "shedding") followed by proteasome-mediated receptor degradation. This translates into inhibition of hepatocyte growth factor/Met-mediated biological activities. However, DN-30 binding to Met also results in partial activation of the Met kinase due to antibody-mediated receptor homodimerization. To safely harness the therapeutic potential of DN-30, its shedding activity must be disassociated from its agonistic activity. Here we show that the DN-30 Fab fragment maintains high affinity Met binding, elicits efficient receptor shedding and down-regulation, and does not promote kinase activation. In Met-addicted tumor cell lines, DN-30 Fab displays potent cytostatic and cytotoxic activity in a dose-dependent fashion. DN-30 Fab also inhibits anchorage-independent growth of several tumor cell lines. In mouse tumorigenesis assays using Met-addicted carcinoma cells, intratumor administration of DN-30 Fab or systemic delivery of a chemically stabilized form of the same molecule results in reduction of Met phosphorylation and inhibition of tumor growth. These data provide proof of concept that monovalency unleashes the full therapeutic potential of the DN-30 antibody and point at DN-30 Fab as a promising tool for Met-targeted therapy.

Fusion between cancer cells and macrophages occurs in a murine model of spontaneous neu+ breast cancer without increasing its metastatic potential.

Cell fusion between neoplastic and normal cells has been suggested to play a role in the acquisition of a malignant phenotype. Several studies have pointed to the macrophage as the normal partner in this fusion, suggesting that the fused cells could acquire new invasive properties and become able to disseminate to distant organs. However, this conclusion is mainly based on studies with transplantable cell lines. We tested the occurrence of cell fusion in the MMTV-neu model of mouse mammary carcinoma. In the first approach, we generated aggregation chimeras between GFP/neu and RFP/neu embryos. Tumor cells would display both fluorescent proteins only if cell fusion with normal cells occurred. In addition, if cell fusion conferred a growth/dissemination advantage, cells with both markers should be detectable in lung metastases at increased frequency. We confirmed that fused cells are present at low but consistent levels in primary neoplasms and that the macrophage is the normal partner in the fusion events. Similar results were obtained using a second approach in which bone marrow from mice carrying the Cre transgene was transplanted into MMTV-neu/LoxP-tdTomato transgenic animals, in which the Tomato gene is activated only in the presence of CRE recombinase. However, no fused cells were detected in lung metastases in either model. We conclude that fusion between macrophages and tumor cells does not confer a selective advantage in our spontaneous model of breast cancer, although these data do not rule out a possible role in models in which an inflammation environment is prominent.

Intestinal microbiota sustains inflammation and autoimmunity induced by hypomorphic RAG defects.

Omenn syndrome (OS) is caused by hypomorphic Rag mutations and characterized by a profound immunodeficiency associated with autoimmune-like manifestations. Both in humans and mice, OS is mediated by oligoclonal activated T and B cells. The role of microbial signals in disease pathogenesis is debated. Here, we show that Rag2(R229Q) knock-in mice developed an inflammatory bowel disease affecting both the small bowel and colon. Lymphocytes were sufficient for disease induction, as intestinal CD4 T cells with a Th1/Th17 phenotype reproduced the pathological picture when transplanted into immunocompromised hosts. Moreover, oral tolerance was impaired in Rag2(R229Q) mice, and transfer of wild-type (WT) regulatory T cells ameliorated bowel inflammation. Mucosal immunoglobulin A (IgA) deficiency in the gut resulted in enhanced absorption of microbial products and altered composition of commensal communities. The Rag2(R229Q) microbiota further contributed to the immunopathology because its transplant into WT recipients promoted Th1/Th17 immune response. Consistently, long-term dosing of broad-spectrum antibiotics (ABXs) in Rag2(R229Q) mice ameliorated intestinal and systemic autoimmunity by diminishing the frequency of mucosal and circulating gut-tropic CCR9(+) Th1 and Th17 T cells. Remarkably, serum hyper-IgE, a hallmark of the disease, was also normalized by ABX treatment. These results indicate that intestinal microbes may play a critical role in the distinctive immune dysregulation of OS.

Targeted therapy by gene transfer of a monovalent antibody fragment against the Met oncogenic receptor.

UNLABELLED: Due to the key role played in critical sub-populations, Met is considered a relevant therapeutic target for glioblastoma multiforme and lung cancers. The anti-Met DN30 antibody, engineered to a monovalent Fab (Mv-DN30), proved to be a potent antagonist, inducing physical removal of Met receptor from the cell surface. In this study, we designed a gene therapy approach, challenging Mv-DN30 in preclinical models of Met-driven human glioblastoma and lung carcinoma. Mv-DN30 was delivered by a Tet-inducible-bidirectional lentiviral vector. Gene therapy solved the limitations dictated by the short half-life of the low molecular weight form of the antibody. In vitro, upon doxycycline induction, the transgene: (1) drove synthesis and secretion of the correctly assembled Mv-DN30; (2) triggered the displacement of Met receptor from the surface of target cancer cells; (3) suppressed the Met-mediated invasive growth phenotype. Induction of transgene expression in cancer cells-transplanted either subcutaneously or orthotopically in nude mice-resulted in inhibition of tumor growth. Direct Mv-DN30 gene transfer in nude mice, intra-tumor or systemic, was followed by a therapeutic response. These results provide proof of concept for a gene transfer immunotherapy strategy by a Fab fragment and encourage clinical studies targeting Met-driven cancers with Mv-DN30. KEY MESSAGE: Gene transfer allows the continuous in vivo production of therapeutic Fab fragments. Mv-DN30 is an excellent tool for the treatment of Met-driven cancers. Mv-DN30 gene therapy represents an innovative route for Met targeting.

ATAD2 is a novel cofactor for MYC, overexpressed and amplified in aggressive tumors.

The E2F and MYC transcription factors are critical regulators of cell proliferation and contribute to the development of human cancers. Here, we report on the identification of a novel E2F target gene, ATAD2, the predicted protein product of which contains both a bromodomain and an ATPase domain. The pRB-E2F pathway regulates ATAD2 expression, which is limiting for the entry into the S phase of the cell cycle. We show that ATAD2 binds the MYC oncogene and stimulates its transcriptional activity. ATAD2 maps to chromosome 8q24, 4.3 Mb distal to MYC, in a region that is frequently found amplified in cancer. Consistent with this, we show that ATAD2 expression is high in several human tumors and that the expression levels correlate with clinical outcome of breast cancer patients. We suggest that ATAD2 links the E2F and MYC pathways and contributes to the development of aggressive cancer through the enhancement of MYC-dependent transcription.

"Active" cancer immunotherapy by anti-Met antibody gene transfer.

Gene therapy provides a still poorly explored opportunity to treat cancer by "active" immunotherapy as it enables the transfer of genes encoding antibodies directed against specific oncogenic proteins. By a bidirectional lentiviral vector, we transferred the cDNA encoding the heavy and light chains of a monoclonal anti-Met antibody (DN-30) to epithelial cancer cells. In vitro, the transduced cells synthesized and secreted correctly assembled antibodies with the expected high affinity, inducing down-regulation of the Met receptor and strong inhibition of the invasive growth response. The inhibitory activity resulted (a) from the interference of the antibody with the Met receptor intracellular processing ("cell autonomous activity," in cis) and (b) from the antibody-induced cleavage of Met expressed at the cell surface ("bystander effect," in trans). The monoclonal antibody gene transferred into live animals by systemic administration or by local intratumor delivery resulted in substantial inhibition of tumor growth. These data provide proof of concept both for targeting the Met receptor and for a gene transfer-based immunotherapy strategy.