MAIN RESULTS

· Hypoxia regulation of the chemokine system. First demonstration that oxygen availability is a determinant parameter in the setting of chemotactic responsiveness to stromal-derived factor 1 (CXCL12). We showed that low oxygen concentration induces high functional expression of the CXCL12 receptor, CXC receptor 4 (CXCR4), in different cell types (monocytes, monocyte-derived macrophages, tumor-associated macrophages, endothelial cells, and cancer cell). The CXCR4 induction by hypoxia is dependent on activation of the hypoxia-inducible factor 1 alpha (HIF-1). We proposed that, in a multistep navigation process, the Hypoxia/HIF-1/CXCR4 pathway may regulate trafficking in and out of hypoxic tissue microenvironments. We also demonstrated that genetic alterations of the von Hippel–Lindau tumor suppressor protein (VHL) is are associated with increased levels of HIF-1 activity, CXCR4 expression and the metastatic potential of cancer cells.

 

· Functional uncoupling of DC functions under hypoxic conditions. Demonstration that low oxygen tension (hypoxia) inhibits expression of several differentiation and maturation markers by dendritic cells (DC) in response to different TLR ligands, as well as their stimulatory capacity for T-cell functions. These events are paralleled by impaired up-regulation of the chemokine receptor CCR7 and enhanced  production of proinflammatory cytokines. In vivo, hypoxia promotes defective DC homing to draining lymph nodes, but enhanced leukocyte recruitment at the site of injection. Thus, hypoxia uncouples the promotion of inflammatory and tissue repair from sentinel functions in DCs.

 

· Tumor-dependent diversion of macrophage function . We reported that tumor-associated macrophages (TAM) from experimental model (fibrosarcoma, melanoma) and human tumors (ovarian carcinoma) display defective production of IL-12, associated with lack of p50/p65 NF-B activation. This phenotype is mediated by high autocrine production of the immunosuppressive cytokine IL-10. We proposed that blocking IL-10 may complement therapeutic strategies aimed at activating type I antitumor immune responses.

 

· Tumor-associated macrophages express a distinct and unique M2-transcriptional program.We generated the first transcriptome profile of TAMs isolated from a murine fibrosarcoma in comparison with peritoneal macrophages (PECs) and myeloid-derived suppressor cells (MDSC). Lipopolysaccharide (LPS)-mediated activation of TAMs resulted in defective expression of several proinflammatory cytokines (eg, IL-1beta, IL-6, TNF-alpha) and chemokines (eg, CCL3), as opposed to a strong up-regulation of immunosuppressive cytokines (IL-10, TGFbeta) and IFN-inducible chemokines (CCL5, CXCL9, CXCL10, CXCL16). Thus, profiling of TAMs from a murine sarcoma revealed unexpected expression of IFN-inducible chemokines, associated with an M2 phenotype (IL-10high, IL-12low), and divergent regulation of the NF-kappaB versus the IRF-3/STAT1 pathway.

 

· p50 NF- α B overexpression in TAM inhibits M1-inflammation and antitumor resistance. We demonstrated that defective responsiveness of TAM from a murine fibrosarcoma and human ovarian carcinoma to M1 activation signals is associated with a massive nuclear localization of the p50 NF-B inhibitory homodimer. p50 overexpression inhibited IL-12 expression in normal macrophages. TAM isolated from p50(-/-) experimental models showed normal production of M1 cytokines, associated with reduced growth of transplanted tumors. Bone marrow chimeras showed that p50 inactivation in hematopoietic cells was sufficient to result in reduced tumor growth.

MAIN OBJECTIVES AND RESEARCH LINES 

Clinical and experimental evidence suggest that tumors require a constant influx of myelomonocytic cells to support the angiogenesis and stroma remodeling needed for their growth. This is mediated by tumor-derived factors, which cause sustained myelopoiesis and the accumulation and functional differentiation of these cells, most of which are tumour-associated macrophages (TAM). We provided evidence for a ‘switch’ in myeloid cell phenotypes, macrophages in particular, during the course of tumour progression. Whereas the functions of classically-activated, ‘M1’ macrophages, during chronic inflammation predisposes a given tissue to tumour initiation, in established tumours macrophages exhibit mainly the alternatively activated ‘M2’ phenotype and are engaged in immunosuppression and the promotion of tumour angiogenesis and metastasis. In conjunction with TAM, Myeloid-Derived Suppressor Cells (MDSC) express an M2-like profile and accumulate in secondary lymphoid organs to promote T cells dysfunction and immune suppression. Based on these premises, main objectives and research lines of our group aim to investigate key tumour-derived molecules/mechanisms supporting differentiation of M2 myeloid populations, TAM and MDSC, to identify new targets suitable for innovative therapeutic intervention.

 

Molecular basis of TAM and MDSC functional polarization . We observed that during tumour progression myeloid cells (TAM and MDSC) express increasing nuclear levels of the p50 NF-B inhibitory homodimer, which correlates with the expression of M2-polarized functions supporting tumour growth. By using the MN/MCA1 fibrosarcoma murine tumour, we will be extend this analysis on other members of the NF-B family (eg. p50, p52, p65, cRel, RelB, IB, IB, IB, Bcl3). In addition, pathways (eg. IL-4, IL-13, IL-10, PGE2) and transcriptional events (eg. STAT3, STAT6, c-maf) promoting the alternative or M2 activation of macrophages we will be investigated. Selected targets will be silenced to investigate their functional significance. This approach involves the use of Lentivirus (LVs) to transduce bone marrow (BM)-derived hematopoietic stem cells (HSCs), which are then transplanted into irradiated experimental models, to monitor both tumour progression and development of polarized inflammatory responses. Identification of determinant M1/M2 polarization markers in murine systems will be next validated by immunohistochemistry in specimens from cancer patients.

 

Role of selected M1/M2-associated molecules in the functional sculpture of the tumor microenvironment . The molecular cross talk between TAM and MDSC and tumour cells will be investigated in vitro by co-culturing these cells types in 3D tumour masses of 700-900 uM diameter, called tumour spheroids, that are generated in non-adherent cultures of experimental model or human tumour cell lines. Cells (macrophages) co-cultured with spheroids infiltrate them over a 6-24h period and are exposed to hypoxia which forms at the centre of these structures due to limited diffusion of oxygen into this region.  Silencing of selected molecules will help to understand their role in the organization of the tumor microenvironment.

 

Role of cancer-related NF-B activity in polarized inflammation. This study aims to clarify important aspects of the functional cross-talk between epithelial cells at various stages of the malignant process and the immune system, looking at mechanisms of activation and diversion of TAM and MDSC functions. In this regard, hepatocyte NF-B activation will be investigated in vivo as a possible event involved in the polarization of TAM and MDSC functions. We will use mdr2 deficient experimental models, a model of spontaneous liver carcinogenesis, that have been engineered so that NF-B activity can be blocked specifically in hepatocytes under control of the antibiotic doxycyclin. TAM and MDSC will be isolated during different steps of tumour development and characterized in terms of transcriptional profile, as well as polarization markers (eg: M1-IL-12, TNF-, IL-1 versus M2: IL-10, TGF, mannose receptor, scavenger receptor, Dectin-1, Fizz) ¹¹, cytokine production (eg. IL-12, TNF-, IL-1, IL-10, TGF-, VEGF, CXCL10).

 

Role of p50 NF-B in the functional phenotype of myeloid cell populations (TAM and MDSC). We will use the Cre-LoxP transgenic models that have either macrophage-specific (CSF-1R-targeted) or MDSC-specific (Ly6G-targeted) p50 NF-B gene ablation. The choice of LY6G to target the MDSC population is due to its more restriced expression in this myeloid/granulocytic, as compared to the granulocyte-specific Gr1 antigen. These models will be crossed with mdr2 deficient models, a model of spontaneous liver carcinogenesis provided by Dr. Eli Pikarsky (Hadassam University, Jerusalem). This study will provide information on the actual role of p50 in promoting the protumoral phenotype of TAM and MDSC. In addition, in vivo evidence will be generated to validate this molecule as potential therapeutic target.

 

Role of p50 NF-B in dendritic cells functions. In order to evaluate whether the tolerogenic action of p50 NF-B  may target other compartments of the immune system, we are characterizing the role of p50 NF-κB in dendritic cells (DC) functions. Our preliminary data show that p50 NF-B plays a non redundant role in DC survival and APC functions. This correlates with the enhanced capability of p50-/- DC to activate T cell responses, in vitro and in vivo. Future efforts will aim to clarify whether targeting p50 NF-B activity may represent a strategy to enhance selective functions of DC, with potential application in vaccination strategies.

 

Role of the IRF-3/STAT1 pathway in tumor-associated inflammation (TAM and MDSC). By investigating the molecular basis driving M2 polarization of TAM, we have recently demonstrated that in parallel to the observed nuclear accumulation of p50 NF-B, these cells express higher activation of the IRF-3/STAT1 pathway, leading to high expression of Intereron-dependent chemokines (CXCL9, CXCL10, CXCL16). To clarify the role of this pathway in driving myeloid cells protumoral functions, we will use IRF-3 deficient models. Our preliminary data indicate that lack of IRF-3 in these models results in altered myeloid cell trafficking during tumour progression and reduced tumour growth (fibrosarcoma and melanoma).

 

Role of p50 NF-B in both endotoxin-tolerance and M2 (alternative) macrophage polarizatio n. Accumulation of p50 homodimers has been observed in endotoxin tolerant macrophages, as well as in TAM. This observation suggests possible similarities between these two events. Mechanistic analysis of the p50 NF-B activity will be performed in LPS-tolerant macrophages versus TAM. Preliminary data, indicate that silencing of p50 NF-B activity prevents development of both M2-polarization of macrophages, as well as their tolerance to LPS.

 

Effects of tumour hypoxia on myelomonocytic cell functions. It is still largely unclear how hypoxia contributes to the organization and functional architecture of the tumour microenvironment. TAM accumulate preferentially in the poorly vascularized region of tumors which are characterized by low oxygen tension and we have previously identified the hypoxia/HIF-1/CXCR4 pathway as a relevant circuit in the tuning of the chemokine system in different cell types (monocytes/macrophages, endothelial cells and cancer cells). More recently, expression of high level of HIF-2 has been reported in TAM. By using the Cre-LoxP transgenic models that have the macrophage-specific (lisozyme-targeted) or MDSC-specific (Ly6G-targeted) HIF-2 gene ablation, we wish to investigate its role in the accumulation and differentiation of myeloid-associated tumor cells.