Emanuela Ognio

Endogenous IL-12 triggers an antiangiogenic program in melanoma cells

The IL12RB2 gene acts as a tumor suppressor in human B cell malignancies. Indeed, Il12rb2 knockout (KO) mice develop spontaneously B cell tumors, but also lung epithelial tumors. This latter phenotype may be related to (i) impairment of host IL-12-mediated immunosurveillance and/or (ii) IL-12 inability to inhibit directly the growth of IL-12 unresponsive malignant cells. To address this issue, we transplanted IL-12R+ B16 melanoma cells into syngeneic Il12rb2 KO mice with the following rationale: (i) these mice have severe defects in IFN-γ production, as well as in cytotoxic T lymphocyte and natural killer cell cytotoxicity, and (ii) they produce but do not use IL-12 that can potentially bind to and target tumor cells only. Il12rb2 KO mice displayed higher endogenous serum levels of IL-12 and developed smaller B16 tumors than WT animals. These tumors showed reduced proliferation, increased apoptosis, and defective microvessel formation related to down-regulated expression of a set of proangiogenic genes previously unrelated to IL-12. Such effects depended on direct activity of endogenous IL-12 on tumor cells in KO mice, and hydrodynamic delivered IL-12 caused further reduced tumorigenicity of B16 cells in these mice. A previously undescribed mechanism of the IL-12 antitumor activity has been here identified and characterized.

Interleukin-27 Inhibits the Growth of Pediatric Acute Myeloid Leukemia in NOD/SCID/Il2rg−/− Mice

Purpose: Acute myeloid leukemia (AML) accounts for more than half of fatal cases in all pediatric leukemia patients; this observation highlights the need of more effective therapies. Thus, we investigated whether interleukin (IL)-27, an immunomodulatory cytokine, functions as an antitumor agent against pediatric AML cells. Experimental Design: Expression of WSX-1 and gp130 on AML cells from 16 pediatric patients was studied by flow cytometry. Modulation of leukemia cell proliferation or apoptosis upon IL-27 treatment in vitro was tested by bromodeoxyuridine/propidium iodide (PI) and Ki67, or Annexin V/PI staining and flow cytometric analysis. The angiogenic potential of AML cells treated or not with IL-27 was studied by chorioallantoic membrane assay and PCR array. In vivo studies were carried out using nonobese diabetic/severe combined immunodeficient (NOD/SCID)/Il2rg−/− mice injected intravenously with five pediatric AML cell samples. Leukemic cells engrafted in PBS and IL-27–treated animals were studied by immunohistochemical/morphologic analysis and by PCR array for expression angiogenic/dissemination-related genes. Results: We provided the first demonstration that (i) AML cells injected into NOD/SCID/Il2rg−/− mice gave rise to leukemia dissemination that was severely hampered by IL-27, (ii) compared with controls, leukemia cells harvested from IL-27–treated mice showed significant reduction of their angiogenic and spreading related genes, and (iii) similarly to what was observed in vivo, IL-27 reduced in vitro AML cell proliferation and modulated the expression of different genes involved in the angiogenic/spreading process. Conclusion: These results provide an experimental rationale for the development of future clinical trials aimed at evaluating the toxicity and efficacy of IL-27. Clin Cancer Res; 18(6); 1630–40. ©2012 AACR.

Complementary IL-23 and IL-27 anti-tumor activities cause strong inhibition of human follicular and diffuse large B-cell lymphoma growth in vivo

Interleukin (IL)-23 and IL-27 are pro-inflammatory cytokines that share functional and structural similarities and may exert anti-tumor activities against solid and hematological malignancies. Here, we asked whether IL-23 and IL-27, alone or in combination, may act directly against human follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) cells. In this study, we demonstrated for the first time that human primary FL and DLBCL cells expressed complete and functional IL-23 and IL-27 receptors (R) and that IL-23 and IL-27 exerted anti-tumor activities in vitro and in vivo through different and complementary mechanisms. In vivo studies using severe combined immunodeficiency /non-obese diabetic mice-injected subcutaneously with human SU-DHL-4 cell line revealed that IL-23 inhibited directly tumor-cell proliferation, whereas IL-27 impaired the angiogenic program of lymphoma cells resulting in strong reduction of cell growth. In addition, combined treatment of IL-23 and IL-27 amplified the anti-tumor effects in vivo as compared with administration of each cytokine alone. These anti-tumor mechanisms were confirmed by in vitro experiments performed with primary lymphoma cells and cell lines. Our results strongly encourage the development of future clinical trials to evaluate the toxicity and efficacy of the IL-23 and IL-27 in lymphoma patients.

Direct inhibition of human acute myeloid leukemia cell growth by IL-12

Acute myeloid leukemia is a haematopoietic malignancy originating from the transformation of myeloid progenitors that proliferate and accumulate in the bone marrow. In AML patients the survival rate at 5 years is 40-50% highlighting the need for novel therapies. In this study we have asked whether IL-12, an immuno-modulatory cytokine with anti-tumor activity, may inhibit directly AML cell growth. We show that the human AML cell lines U937, K562 and THP-1 expressed both chains of the IL-12 receptor (R), i.e. IL-12Rβ1 and IL-12Rβ2. IL-12 inhibited the angiogenic potential of AML cells in vitro, but did not affect their survival or proliferation. In vivo experiments were performed using SCID-NOD mice injected intraperitoneally (i.p.) with the human U937 AML cell line and subsequently treated with human recombinant IL-12 or PBS i.p. Histological, immunohistochemical and flow cytometric analyses on explanted tumors revealed that IL-12 reduced new vessel formation, induced apoptosis and inhibited tumor cell proliferation. Studies on a panel of angiogenesis related genes in explanted tumors using PCR arrays showed significantly down-regulated expression of numerous pro-angiogenic genes including VEGF-C, IL-6, IL-8, CXCL1, CXCL6 and alanyl aminopeptidase in IL-12 vs PBS treated mice. This study shows for the first time that IL-12 targets directly AML cell growth and paves the way to further investigation of IL-12 as potential drug for AML treatment.

A novel liposomal Clodronate depletes tumor-associated macrophages in primary and metastatic melanoma: Anti-angiogenic and anti-tumor effects

Abstract The depletion of tumor-associated macrophages (TAMs), involved in different stages of cancer development and progression, is an appealing strategy in cancer therapy. We developed novel Clodronate-containing liposomes (Clo-Lipo-DOTAP) presenting physicochemical properties (size distribution, polydispersity index and Z-potential) suited for safe storage and injections. In vitro, Clo-Lipo-DOTAP inhibited proliferation, reduced viability and induced apoptosis of a macrophage-like cell line in a dose- and time-dependent manner. In proof of functionality experiments, Clo-Lipo-DOTAP depleted macrophages in a genetic mouse model of chronic hepatitis and hepatocellular carcinoma leading to a significant reduction of F4/80-positive cells in the liver and spleen of treated mice compared to PBS-treated controls. The number of granulocytes, B and T lymphocytes was not affected. In B16/F10 subcutaneous melanoma-bearing mice, Clo-Lipo-DOTAP significantly reduced the volume of primary tumors (Pxa0 In conclusion, the depletion of TAMs in primary and metastatic melanoma presents anti-tumor efficacy via inhibition of angiogenesis and modulation of inflammation related cytokines.

Absence of IL-12Rβ2 in CD33 + CD38 + pediatric acute myeloid leukemia cells favours progression in NOD/SCID/IL2RγC-deficient mice

Childhood acute myeloid leukemia (AML) is a hematological malignancy in which tumor burden is continuously replenished by leukemic-initiating cells (ICs), which proliferate slowly and are refractory to chemotherapeutic agents. We investigated whether interleukin (IL)-12, an immuno-modulatory cytokine with anti-tumor activity, may target AML blasts (CD45+CD33+) and populations known to contain leukemia ICs (that is, CD34+CD38−, CD33+CD38+ and CD44+CD38− cells). We demonstrate for the first time that: i) AML blasts and their CD34+CD38−, CD33+CD38+, CD44+CD38− subsets express the heterodimeric IL-12 receptor (IL-12R), ii) AML cells injected subcutaneously into NOD/SCID/Il2rg−/− (NSG) mice developed a localized tumor mass containing leukemic ICs and blasts that were virtually eliminated by IL-12 treatment, iii) AML cells injected intravenously into NSG mice engrafted within the first month in the spleen, but not in bone marrow or peripheral blood. At this time, IL-12 dramatically dampened AML CD45+CD33+, CD34+CD38−, CD33+CD38+ and CD44+CD38− populations, only sparing residual CD33+CD38+ cells that did not express IL-12Rβ2. From 30 to 60 days after the initial inoculum, these IL-12-unresponsive cells expanded and metastasized in both control and IL-12-treated NSG mice. Our data indicate that the absence of IL-12Rβ2 in pediatric AML cells favours leukemia progression in NOD/SCID/IL2Rγc-deficient mice.

Interleukin-17A promotes the growth of human germinal center derived non-Hodgkin B cell lymphoma

Interleukin (IL)-17A belongs to IL-17 superfamily and binds the heterodimeric IL-17 receptor (R)(IL-17RA/IL-17RC). IL-17A promotes germinal center (GC) formation in mouse models of autoimmune or infectious diseases, but the role of IL-17A/IL-17AR complex in human neoplastic GC is unknown. In this study, we investigated expression and function of IL-17A/IL-17AR in the microenvironments of 44 B cell non-Hodgkin lymphomas (B-NHL) of GC origin (15 follicular lymphomas, 17 diffuse large B cells lymphomas and 12 Burkitt lymphomas) and 12 human tonsil GC. Furthermore, we investigated the role of IL-17A in two in vivo models of GC B cell lymphoma, generated by s.c. injection of SU-DHL-4 and OCI-Ly8 cell lines in Severe combined immunodeficiency (SCID)/Non Obese Diabetic (NOD) mice. We found that: (i) B-NHL cell fractions and tonsil GC B cells expressed IL-17RA/IL-17RC, (ii) IL-17A signaled in both cell types through NF-kBp65, but not p38, ERK-1/2, Akt or NF-kBp50/105, phosphorylation, (iii) IL-17A was expressed in T cells and mast cells from neoplastic and normal GC microenvironments, (iv) IL-17A rendered tonsil GC B cells competent to migrate to CXCL12 and CXCL13 by downregulating RGS16 expression; (v) IL-17A stimulated in vitro proliferation of primary B-NHL cells; (vi) IL-17A (1 μg/mouse-per dose) stimulated B-NHL growth in two in vivo models by enhancing tumor cell proliferation and neo-angiogenesis. This latter effect depended on IL-17A-mediated induction of pro-angiogenic gene expression in tumor cells and direct stimulation of endothelial cells. These data define a previously unrecognized role of human IL-17A in promoting growth of GC-derived B-NHL and modulating normal GC B cell trafficking.

Androgens affect muscle, motor neuron, and survival in a mouse model of SOD1-related amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective loss of upper and lower motor neurons and skeletal muscle atrophy. Epidemiologic and experimental evidence suggest the involvement of androgens in ALS pathogenesis, but the mechanism through which androgens modify the ALS phenotype is unknown. Here, we show that androgen ablation by surgical castration extends survival and disease duration of a transgenic mouse model of ALS expressing mutant human SOD1 (hSOD1-G93A). Furthermore, long-term treatment of orchiectomized hSOD1-G93A mice with nandrolone decanoate (ND), an anabolic androgenic steroid, worsened disease manifestations. ND treatment induced muscle fiber hypertrophy but caused motor neuron death. ND negatively affected survival, thereby dissociating skeletal muscle pathology from life span in this ALS mouse model. Interestingly, orchiectomy decreased androgen receptor levels in the spinal cord and muscle, whereas ND treatment had the opposite effect. Notably, stimulation with ND promoted the recruitment of endogenous androgen receptor into biochemical complexes that were insoluble in sodium dodecyl sulfate, a finding consistent with protein aggregation. Overall, our results shed light on the role of androgens as modifiers of ALS pathogenesis via dysregulation of androgen receptor homeostasis.

Enhanced anti-neuroblastoma activity of a fenretinide complexed form after intravenous administration

Objectivesu2002 The major limitation to successful chemotherapy of neuroblastoma (NB) is the toxicity and the poor bioavailability of traditional drugs.

IL-25 dampens the growth of human germinal center-derived B-cell non Hodgkin Lymphoma by curtailing neoangiogenesis

ABSTRACT Interleukin (IL)-25, a member of the IL-17 cytokine superfamily, is produced by immune and non-immune cells and exerts type 2 pro-inflammatory effects in vitro and in vivo. The IL-25 receptor(R) is composed of the IL-17RA/IL-17RB subunits. Previous work showed that germinal centre (GC)-derived B-cell non Hodgkin lymphomas (B-NHL) expressed IL-17AR, formed by IL-17RA and IL-17RC subunits, and IL-17A/IL-17AR axis promoted B-NHL growth by stimulating neoangiogenesis. Here, we have investigated expression and function of IL-25/IL-25R axis in lymph nodes from human GC-derived B-NHL, i.e. Follicular Lymphoma (FL,10 cases), Diffuse Large B Cell Lymphoma (6 cases) and Burkitt Lymphoma (3 cases). Tumor cells expressed IL-25R and IL-25 that was detected also in non-malignant cells by flow cytometry. Immunohistochemical studies confirmed expression of IL-25R and IL-25 in FL cells, and highlighted IL-25 expression in bystander elements of the FL microenvironment. IL-25 i) up-regulated phosphorylation of NFkBp65, STAT-1 and JNK in B-NHL cells; ii) inhibited in vitro proliferation of the latter cells; iii) exerted anti-tumor activity in two in vivo B-NHL models by dampening expression of pro-angiogenic molecules as VEGF-C, CXCL6 and ANGPT3. In conclusion, IL-25, that is intrinsically pro-angiogenic, inhibits B-NHL growth by reprogramming the angiogenic phenotype of B-NHL cells.