Alessia Zorzoli

γδ T-cell reconstitution after HLA-haploidentical hematopoietic transplantation depleted of TCR-αβ+/CD19+ lymphocytes

We prospectively assessed functional and phenotypic characteristics of γδ T lymphocytes up to 7 months after HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) depleted of αβ(+) T cells and CD19(+) B cells in 27 children with either malignant or nonmalignant disorders. We demonstrate that (1) γδ T cells are the predominant T-cell population in patients during the first weeks after transplantation, being mainly, albeit not only, derived from cells infused with the graft and expanding in vivo; (2) central-memory cells predominated very early posttransplantation for both Vδ1 and Vδ2 subsets; (3) Vδ1 cells are specifically expanded in patients experiencing cytomegalovirus reactivation and are more cytotoxic compared with those of children who did not experience reactivation; (4) these subsets display a cytotoxic phenotype and degranulate when challenged with primary acute myeloid and lymphoid leukemia blasts; and (5) Vδ2 cells are expanded in vitro after exposure to zoledronic acid (ZOL) and efficiently lyse primary lymphoid and myeloid blasts. This is the first detailed characterization of γδ T cells emerging in peripheral blood of children after CD19(+) B-cell and αβ(+) T-cell-depleted haplo-HSCT. Our results can be instrumental to the development of clinical trials using ZOL for improving γδ T-cell killing capacity against leukemia cells. This trial was registered at www.clinicaltrials.gov as #NCT01810120.

Therapeutic Targeting of TLR9 Inhibits Cell Growth and Induces Apoptosis in Neuroblastoma

The Toll-like receptor 9 (TLR9) evolved to cope with pathogens, but it is expressed in a variety of tumors for reasons that are unclear. In this study, we report that neuroblastoma (NB) cells express functional TLR9. Liposome-complexed CpG oligonucleotides inhibited the proliferation of TLR9-expressing NB cells and induced caspase-dependent apoptotic cell death. Inhibitory oligonucleotides (iODNs) abrogated these effects. RNA interference reduced TLR9 expression but not to the level where functional responses to CpG were abolished. Compared with free CpG, liposomal formulations of NB-targeted CpG (TL-CpG) significantly prolonged the survival of mice bearing NB tumor xenografts. While CpG alone lacked antitumor efficacy in NOD/SCID/IL2rg(-/-) mice, TL-CpG retained significant efficacy related to direct effects on tumor cells. TLR9 expression in primary human NB specimens was found to correlate inversely with disease stage. Our findings establish functional expression of TLR9 in NB and suggest that TLR9 may represent a novel theranostic target in this disease.

Neuroblastoma-targeted Nanoparticles Entrapping siRNA Specifically Knockdown ALK

RNA interference molecules have some advantages as cancer therapeutics, including a proved efficacy on both wild-type (WT) and mutated transcripts and an extremely high sequence-specificity. The most significant hurdle to be overcome if exogenous small interfering RNAs (siRNA) is to be used therapeutically is the specific, effective, nontoxic delivery of siRNA to its intracellular site of action. At present, human applications are confined almost exclusively to targets within the liver, where the delivery systems naturally accumulate, and extra-hepatic targets remain a challenge. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that has recently been shown to contribute to the cell growth and progression of human neuroblastoma (NB). We investigated its potential as a therapeutic target in NB by generating anti-GD₂-targeted nanoparticles that carry ALK-directed siRNA, which are specifically and efficiently delivered to GD₂-expressing NB cells. Relative to free ALK-siRNA, anti-GD₂-targeted liposomal formulations of ALK-siRNA had low plasma clearance, increased siRNA stability, and improved binding, uptake, silencing and induction of cell death, and specificity for NB cells. In NB xenografts, intravenous (i.v.) injection of the targeted ALK-siRNA liposomes showed gene-specific antitumor activity with no side effects. ALK-selective siRNA entrapped in anti-GD₂-targeted nanoparticles is a promising new modality for NB treatment.

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.

The use of the orthotopic model to validate antivascular therapies for cancer

The orthotopic model reproduces aspects of the tumour microenvironment and emulates a number of important biological features of cancer progression, angiogenesis, metastasis and resistance. Due to its parallels with human cancer, the model can be used to evaluate therapeutic responses to various therapies. This review outlines the importance of using the orthotopic implantation of tumour cells in mice models for evaluating the effectiveness of antivascular therapies.

Interleukin-30 expression in prostate cancer and its draining lymph nodes correlates with advanced grade and stage.

Purpose: The interleukin (IL)-27 cytokine subunit p28, also called IL-30, has been recognized as a novel immunoregulatory mediator endowed with its own functions. These are currently the subject of discussion in immunology, but completely unexplored in cancer biology. We set out to investigate the role of IL-30 in prostate carcinogenesis and its effects on human prostate cancer (hPCa) cells. Experimental Design: IL-30 expression, as visualized by immunohistochemistry and real-time reverse transcriptase PCR on prostate and draining lymph nodes from 125 patients with prostate cancer, was correlated with clinicopathologic data. IL-30 regulation of hPCa cell viability and expression of selected gene clusters was tested by flow cytometry and PCR array. Results: IL-30, absent in normal prostatic epithelia, was expressed by cancerous epithelia with Gleason ≥ 7% of 21.3% of prostate cancer stage I to III and 40.9% of prostate cancer stage IV. IL-30 expression by tumor infiltrating leukocytes (T-ILK) was higher in stage IV that in stage I to III prostate cancer (P = 0.0006) or in control tissue (P = 0.0011). IL-30 expression in prostate draining lymph nodes (LN)-ILK was higher in stage IV than in stage I to III prostate cancer (P = 0.0031) or in control nodes (P = 0.0023). The main IL-30 sources were identified as CD68+ macrophages, CD33+/CD11b+ myeloid cells, and CD14+ monocytes. In vitro, IL-30 stimulated proliferation of hPCa cells and also downregulated CCL16/LEC, TNFSF14/LIGHT, chemokine-like factor (CKLF), and particularly CKLF-like MARVEL transmembrane domain containing 3 (CMTM3) and greatly upregulated ChemR23/CMKLR. Conclusions: We provide the first evidence that IL-30 is implicated in prostate cancer progression because (i) its expression by prostate cancer or T- and LN-ILK correlates with advanced disease grade and stage; and (ii) IL-30 exerts protumor activity in hPCa cells. Clin Cancer Res; 20(3); 585–94. ©2013 AACR.

Recent Advances in Targeted Anti-Vasculature Therapy: The Neuroblastoma Model

Novel anti-vasculature strategies that are emerging for the treatment of cancer and for the inhibition of angiogenesis may be a promising new tool for the adjuvant therapy of malignant tumours. Over the last fifteen years, several reports have been published concerning the relationship between tumour progression and angiogenesis in experimental models of neuroblastoma in vitro and in vivo. Moreover, a high vascular index in neuroblastoma correlates with poor prognosis, suggesting dependence of aggressive tumour growth on active angiogenesis. Here, we present an overview of the most recent advances in anti-vasculature therapy of neuroblastoma, and describe some preclinical results as well as future perspectives.