Serena Zilio

Chemokine nitration prevents intratumoral infiltration of antigen-specific T cells

Blocking CCL2 nitration in tumors promoted CD8+ influx and reduced tumor growth and prolonged survival in mice when combined with adoptive cell therapy.

Therapeutic targeting of myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSCs) represent a subset of myeloid cells that expand under pathological conditions, such as cancer development, acute and chronic infections, trauma, bone marrow transplantations, and some autoimmune diseases. MDSCs mediate a negative regulation of the immune response by affecting different T lymphocyte subsets. Potential mechanisms, which underlie this inhibitory activity range from those requiring direct cell-to-cell contact with others, more indirect, and mediated by the modification of the microenvironment. Pharmacological inhibition of MDSC suppressive pathways is a promising strategy to overcome disease-induced immune defects, which might be a key step in enhancing the effectiveness of immune-based therapies.

Transcription factors in myeloid-derived suppressor cell recruitment and function.

In normal hematopoiesis, differentiation and maturation of cell populations belonging to various lineages are tightly regulated by the interaction of many transcription factors. The relative numbers of different myeloid cells depends on their proliferative/apoptotic rate, while their identity relates to their recruitment to the sites of action and the expression of specific genes regulating their function. Under pathological conditions, as during chronic inflammation and cancer development, an aberrant hematopoiesis occurs, with the consequent expansion of myeloid-derived suppressor cells (MDSCs). These cells have distinctive properties that determine their ability to tune down the immune system by principally inactivating CD8(+) T cells. Understanding the molecular networks regulating the phenotypic and functional determination of MDSCs is essential to identify potential therapeutic targets to revert immune deregulation in cancer.

Differently immunogenic cancers in mice induce immature myeloid cells that suppress CTL in vitro but not in vivo following transfer

Tumors frequently induce immature myeloid cells (iMC), which suppress specific and unrelated cytotoxic T lymphocyte (CTL) responses and are termed myeloid-derived suppressor cells (MDSC). Mainly analyzed by in vitro assays in tumor transplantation models, little is known about their function in autochthonous tumor models in vivo. We analyzed iMC in 3 SV40 large T (Tag)-driven conditional autochthonous cancer models with different immune status: (1) Early Tag-specific CTL competence and rare stochastic Tag activation leading to sporadic cancer, which induces an aberrant immune response and CTL tolerance; (2) Cre/LoxP recombinase-mediated hepatocellular carcinoma (HCC) development in neonatal Tag-tolerant mice; and (3) Tag-activation through Cre recombinase-encoding viruses in the liver and HCC development with systemic anti-Tag CTL immunity. In the first but not two latter models, tumors induced CTL hyporesponsiveness to tumor-unrelated antigens. Regardless of the model, tumors produced interleukin-6 and vascular endothelial growth factor but not granulocyte macrophage–colony-stimulating factor (GM-CSF) and induced iMC (CD11b(+)Gr-1(int)) that suppressed CTL responses in vitro. None of the iMC from the different tumor models suppressed CTL responses in adoptive cell transfer experiments unless GM-CSF was provided in vivo. Together, iMC expand independent of the type of antitumor response and are not immunosuppressive in a cell-autonomous fashion.

Neutrophils and Granulocytic MDSC: The Janus God of Cancer Immunotherapy.

Neutrophils are the most abundant circulating blood cell type in humans, and are the first white blood cells recruited at the inflammation site where they orchestrate the initial immune response. Although their presence at the tumor site was recognized in the 1970s, until recently these cells have been neglected and considered to play just a neutral role in tumor progression. Indeed, in recent years neutrophils have been recognized to play a dual role in tumor development by either assisting the growth, angiogenesis, invasion, and metastasis or by exerting tumoricidal action directly via the secretion of antitumoral compounds, or indirectly via the orchestration of antitumor immunity. Understanding the biology of these cells and influencing their polarization in the tumor micro- and macro-environment may be the key for the development of new therapeutic strategies, which may finally hold the promise of an effective immunotherapy for cancer.

4PD functionalized dendrimers: A flexible tool for in vivo gene silencing of tumor-educated myeloid cells

Myeloid cells play a key role in tumor progression and metastasis by providing nourishment and immune protection, as well as facilitating cancer invasion and seeding to distal sites. Although advances have been made in understanding the biology of these tumor-educated myeloid cells (TEMCs), their intrinsic plasticity challenges our further understanding of their biology. Indeed, in vitro experiments only mimic the in vivo setting, and current gene-knockout technologies do not allow the simultaneous, temporally controlled, and cell-specific silencing of multiple genes or pathways. In this article, we describe the 4PD nanoplatform, which allows the in vivo preferential transfection and in vivo tracking of TEMCs with the desired RNAs. This platform is based on the conjugation of CD124/IL-4Rα–targeting peptide with G5 PAMAM dendrimers as the loading surface and can convey therapeutic or experimental RNAs of interest. When injected i.v. in mice bearing CT26 colon carcinoma or B16 melanoma, the 4PD nanoparticles predominantly accumulate at the tumor site, transfecting intratumoral myeloid cells. The use of 4PD to deliver a combination of STAT3- and C/EBPβ-specific short hairpin RNA or miR-142-3p confirmed the importance of these genes and microRNAs in TEMC biology and indicates that silencing of both genes is necessary to increase the efficacy of immune interventions. Thus, the 4PD nanoparticle can rapidly and cost effectively modulate and assess the in vivo function of microRNAs and mRNAs in TEMCs.

Abstract 1449: In vivo targeted silencing of CCR1 and CCR5 repolarizes pro-tumoral myeloid cells in retinoblastoma positive neutrophils with a strong anti-tumor activity

Introduction: Depending on their polarization, myeloid cells can either promote or restrain tumor progression. By secreting myelopoietic factors tumours promote myeloid cells polarization toward a phenotype that provides immune protection and that stimulate neoplastic cells growth, invasiveness, and metastasis. Chemokines and their cognate receptors are thought to play a key role in myeloid cell trafficking, however, their study in vivo is hindered by their signal redundancy and integration. Methods: By taking advantage of a newly developed nanoplatform that allows the in vivo preferential transfection of tumor infiltrating myeloid cells (TIMC, aka Myeloid derived suppressor cells, MDSC, and tumor associated macrophages, TAM), we evaluated the effect of CCR-1, CCR-2, CCR-4, CCR-5 and/or CCR-7 silencing on TAMC trafficking, phenotype, and function. Results: While CCR-4 and -7 targeted silencing did not affect tumor progression, simultaneous silencing of CCR-1 and CCR-5 significantly restrained tumor growth and greatly modifies tumor micro-environment. Mechanistic in vivo and in vitro analysis surprisingly indicate that myeloid cell trafficking was not altered whereas myeloid cell polarization was. In particular, CCR-1 and CCR-5 blockade restrained MDSC differentiation and promote the accumulation of retinoblastoma positive, neutrophil-like cells with a strong tumoricidal activity. Discussion: Our data indicate that CCR-1 and CCR-5 engagement (most likely via CCL-3 and CCL-4) is necessary for the pro-tumoral polarization of myeloid cells in cancer. Our findings not only improve our understanding of myeloid cells differentiation in cancer but, also, strongly suggest that targeted inhibition of CCR1 and CCR5 on TIMC can be a new and effective anti-tumor immune-therapeutic strategy that convert pro-tumoral MDSCs into tumoricidal neutrophils. Citation Format: Serena Zilio, Jennifer Vella, Adriana De La Fuente, Vincenzo Bronte, Emilia Mazza, Silvio Bicciato, Paolo Serafini. In vivo targeted silencing of CCR1 and CCR5 repolarizes pro-tumoral myeloid cells in retinoblastoma positive neutrophils with a strong anti-tumor activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1449.

Tumour-Induced Immune Suppression by Myeloid Cells

One of the main, long-lasting questions in tumor immunology concerns the extreme low efficiency of the immune system toward cancer cells. Although tumor cells are syngenic to the host, they are nonetheless a source of potentially immune stimulating antigens; yet, to date, no tumor can be fought without invasive surgery or chemotherapy. Tumor progression is often associated with an increased rate of myelopoiesis and a consequent accumulation of different subsets of myeloid cells in secondary lymphoid organs and tumor sites. These cells, which include regulatory/immature dendritic cells, myeloid-derived suppressor cells, and tumor-associated macrophages, have profound detrimental effects on anti-tumor immune response and might represent the main cause for immunotherapy failure. In this chapter, we outline the main strategies operated by cancer to circumvent the efficacy of immune responses, which involve the release of soluble factors able to alter myelopoietic homeostasis. We further discuss the relationship between different myeloid cells, highlighting the plasticity of the myeloid compartment, and its importance in regulating T lymphocyte responses. A progressive understanding of the main features and properties of myeloid cells in tumor-bearing hosts will allow us to improve the effectiveness of cancer immunotherapy.