ROLE OF CCL2 AND ITS RECEPTORS CCR2 AND D6 IN THE ACTIVATION AND POLARIZATION OF TUMOR-ASSOCIATED MACROPHAGES

Chemokines are well known to play a major role in tumor progression and metastasis. In particular CCL2 is over-expressed in several human cancers and their higher levels correlate with poor prognosis and shorter outcomes. Here we reported two different studies in which CCL2 receptors, the canonical CCR2 and the atypical D6 (or ACKR2) were examined for their involvement in tumor progression. In particular D6 was investigated for its expression and its ability to shape CCL2 gradient in Kaposi\u2019s sarcoma, whereas CCR2 has been analyzed as potential modeler of TAM polarization. D6 is an atypical chemokine receptor acting as a decoy and scavenger for inflammatory CC chemokines expressed in lymphatic endothelial cells. Here, we report that D6 is also expressed by Kaposi\u2019s sarcoma (KS) which is a tumor ontogenetically related to lymphatic endothelium, yet its role in tumor progression was hitherto unknown. D6 expression was evaluated by immunohistochemistry in a cohort of KS patients and its role in cancer progression was investigated in an in vivo KS model. Both in human tumors and in the experimental model, D6 expression levels were inversely correlated with tumor aggressiveness, and directly correlated with increased chemokine-driven infiltration of macrophages and their acquisition of a pro-angiogenic phenotype. Inhibition of monocyte recruitment reduced growth of D6-incompetent tumors, while adoptive transfer of wt but not CCR2-/- macrophages increased the growth rate of D6-competent neoplasms. In the KS model, which presents the B-Raf V600E activating mutation, inhibition of B-Raf or downstream ERK pathway induced D6 expression, and in progressing human KS tumors activation of the K-Ras/B-Raf/ERK pathway correlate with reduced levels of D6 expression. These results indicate that activation of the K-Ras/B-Raf/ERK pathway during KS progression down-regulates D6 expression, which unleashes chemokine-mediated macrophage recruitment and their acquisition of an M2-like phenotype supporting angiogenesis and tumor growth. Thereafter, we wanted to deeper investigate how CCR2 support TAM M2 polarization firstly by using an in vitro system. Wt and CCR2-/- macrophages were polarized with M1 and M2 stimuli and analyzed for gene expression and cytokines production. While no difference was found in M2 polarized macrophages, CCR2-/- M1 or LPS activated macrophages showed higher expression of inflammatory genes and reduced production of the anti-inflammatory cytokine IL-10 and of the pro-angiogenic cytokine VEGF when compared to wt macrophages. The impaired IL-10 production was also confirmed by treating human monocytes with the CCR2 antagonist RS-504393. After LPS stimulation, CCR2-/- macrophages showed reduced activation of NF-kB and p38 MAPK when compared to wt macrophages indicating a cross talk between CCR2 and TLR4 signaling pathways. The contribution of CCR2 to cancer growth was evaluated with a transplantable lung cancer model that grew slower when co-injected with CCR2-/- macrophages, presenting a marked M1 phenotype of infiltrating TAM and a higher number of both CD4+ and CD8+ T cells, correlated with a decreased number of splenic T regulatory cells when compared to wt macrophages holding-tumors. Taken together these data indicate that CCR2 expression by macrophages not only induce their recruitment to tumor site but also affect their polarization and anti-tumor potential

CXCL5-mediated accumulation of mature neutrophils in lung cancer tissues impairs the differentiation program of anticancer CD8 T cells and limits the efficacy of checkpoint inhibitors

Lung tumor-infiltrating neutrophils are known to support growth and dissemination of cancer cells and to suppress T cell responses. However, the precise impact of tissue neutrophils on programming and differentiation of anticancer CD8 T cells in vivo remains poorly understood. Here, we identified cancer cell-autonomous secretion of CXCL5 as sufficient to drive infiltration of mature, protumorigenic neutrophils in a mouse model of non-small cell lung cancer (NSCLC). Consistently, CXCL5 transcripts correlate with neutrophil density and poor prognosis in a large human lung adenocarcinoma compendium. CXCL5 genetic deletion, unlike antibody-mediated depletion, completely and selectively prevented neutrophils accumulation in lung tissues. Depletion of tumor-infiltrating neutrophils promoted expansion of tumor-specific CD8 T cells, differentiation into effector cells and acquisition of cytolytic functions. Transfer of effector CD8 T cells into neutrophil-rich tumors, inhibited IFN-ϒ production, indicating active suppression of effector functions. Importantly, blocking neutrophils infiltration in the lung, overcame resistance to checkpoint blockade. Hence, this study demonstrates that neutrophils curb acquisition of cytolytic functions in lung tumor tissues and suggests targeting of CXCL5 as a strategy to restore anti-tumoral T cell functions

The atypical chemokine receptor 2 promotes breast cancer lung metastatization through modulation of the metastatic niche

.Introduction: The atypical chemokine receptor 2 (ACKR2) is a scavenger receptor for most inflammatory CC chemokines. It plays a protective role in chronic inflammation, autoimmunity and inflammation-related cancer. The aim of this project is to investigate the role of ACKR2 in primary tumor development and metastatization in different mouse model. Materials and methods: NeuT mice (overexpressing the rat oncogene Her2) crossed with ACKR2-/- mice and orthotopic and intravenous injection of the breast carcinoma cell line 4T1 in Wild Type (WT) and ACKR2-/- Balb/c mice. Tumors and metastasis were studied with in vivo imaging systems, immunohistochemistry and RT-PCR analysis; blood composition and metastatic organ infiltrate were studied with FACS analysis. Results: Tumor arising in ACKR2-/- NeuT mice showed a more aggressive phenotype when compared to WT NeuT mice. On the contrary, ACKR2-/- NeuT mice were protected from spontaneous lung metastasis. Absence of ACKR2 was also protective against spontaneous lung metastasis using the 4T1 orthotopic model of breast cancer but not in the intravenous injected model. Cytofluorimetric analysis indicated an increased of Ly6G+ polymorphonuclear cells and Ly6Chigh monocytes in the blood of ACKR2-/- in and in the pre-metastatic lungs with a concomitant decrease of these cells in the bone marrow during tumor progression. Conclusion: These results indicate that ACKR2 expression has a dual but opposite role in breast tumor, inhibiting primary tumor growth but promoting lung metastatization trough the regulation of leukocyte infiltration and activation in the premetastatic niche

An indirect method for assessing the abundance of introduced pest Myocastor coypus (Rodentia) in agricultural landscapes

Pest management requires the development of robust monitoring tools. In Italy, coypu Myocastor coypus (nutria) have been controlled since the early 1990s, but the effectiveness of these measures has never been tested. With the aim of developing a reliable and volunteer-based method for the long-term monitoring of coypu abundance in agricultural landscapes, we calibrated an index based on surveys for coypu paths against density estimates obtained through a standardized mark–recapture technique. Two trapping sessions were performed in winter for each of 12 1-km long stretches of irrigation canals and watercourses using 15 baited cage traps. Trapping sessions lasted 7 days each, with a 10-day break between sessions. Population size was assessed using three methods: Peterson–Lincoln's formula, capwire estimators and accumulation curves. Active coypu paths and five habitat variables were recorded by walking on the edge of both banks. The variables were then related to population size (y) by means of multi-regressive models, testing for the predictive power of the selected models by leave-one-out cross-validation. Multi-regressive models included only the number of coypu paths with the best performances achieved by the model based on Peterson–Lincoln formula, supporting path count as an effective method to assess the abundance of the coypu in agricultural landscapes. Concurrently, to assess the field suitability of the indirect method, surveys for coypu paths were carried out on 122 randomly chosen 3-km long stretches of irrigation canals and watercourses in the central part of the River Po valley (c. 15?000?km2; N Italy). The highest (>8/100?m) mean number of paths was recorded in the central part of the study area. According to the regression models, the overall number of coypu is predicted to range between 350 000 and 1 100 000, raising doubts about the effectiveness of current control measures