The impact of macrophages on endothelial cells is potentiated by cycling hypoxia: Enhanced tumor inflammation and metastasis.

Cycling hypoxia (cyH), neo-angiogenesis, and tumor-associated macrophages are key features of the tumor microenvironment. In this study, we demonstrate that cyH potentiates the induction by unpolarized and M1-like macrophages of endothelial inflammatory phenotype and adhesiveness for monocytes and cancer cells. This process triggers a positive feedback loop sustaining tumor inflammation. This work opens the door for innovative therapeutic strategies to treat tumor inflammation and metastasis. In cancers, the interaction between macrophages and endothelial cells (ECs) regulates tumor inflammation and metastasis. These cells are both affected by cycling hypoxia (cyH), also called intermittent hypoxia, a feature of the tumor microenvironment. cyH is also known to favor tumor inflammation and metastasis. Nonetheless, the potential impact of cyH on the dialog between macrophages and ECs is still unknown. In this work, the effects of unpolarized, M1-like, and M2-like macrophages exposed to normoxia, chronic hypoxia (chH), and cyH on endothelial adhesion molecule expression, pro-inflammatory gene expression, and EC adhesiveness for monocytes and cancer cells were investigated. cyH increased the ability of unpolarized and M1-like macrophages to induce EC inflammation and to increase the expression of the EC endothelial adhesion molecule ICAM1, respectively. Unpolarized, M1-like, and M2-like macrophages were all able to promote EC adhesive properties toward cancer cells. Furthermore, the ability of macrophages (mostly M1-like) to shift EC phenotype toward one allowing cancer cell and monocyte adhesion onto ECs was potentiated by cyH. These effects were specific to cyH because they were not observed with chH. Together, these results show that cyH amplifies the effects of macrophages on ECs, which may promote tumor inflammation and metastasis.

A bi-directional dialog between vascular cells and monocytes/macrophages regulates tumor progression.

Cancer progression largely depends on tumor blood vessels as well on immune cell infiltration. In various tumors, vascular cells, namely endothelial cells (ECs) and pericytes, strongly regulate leukocyte infiltration into tumors and immune cell activation, hence the immune response to cancers. Recently, a lot of compelling studies unraveled the molecular mechanisms by which tumor vascular cells regulate monocyte and tumor-associated macrophage (TAM) recruitment and phenotype, and consequently tumor progression. Reciprocally, TAMs and monocytes strongly modulate tumor blood vessel and tumor lymphatic vessel formation by exerting pro-angiogenic and lymphangiogenic effects, respectively. Finally, the interaction between monocytes/TAMs and vascular cells is also impacting several steps of the spread of cancer cells throughout the body, a process called metastasis. In this review, the impact of the bi-directional dialog between blood vascular cells and monocytes/TAMs in the regulation of tumor progression is discussed. All together, these data led to the design of combinations of anti-angiogenic and immunotherapy targeting TAMs/monocyte whose effects are briefly discussed in the last part of this review.

Cycling hypoxia promotes a pro-inflammatory phenotype in macrophages via JNK/p65 signaling pathway.

Cycling hypoxia (cyH), also called intermittent hypoxia, occurs in solid tumors and affects different cell types in the tumor microenvironment and in particular the tumor-associated macrophages (TAMs). As cyH and TAMs both favor tumor progression, we investigated whether cyH could drive the pro-tumoral phenotype of macrophages. Here, the effects of cyH on human THP-1 macrophages and murine bone marrow-derived macrophages (BMDM), either unpolarized M0, or polarized in M1 or M2 phenotype were studied. In M0 macrophages, cyH induced a pro-inflammatory phenotype characterized by an increase in TNFα and IL-8/MIP-2 secretion. CyH amplified the pro-inflammatory phenotype of M1 macrophages evidenced by an increased pro-inflammatory cytokine secretion and pro-inflammatory gene expression. Furthermore, cyH increased c-jun activation in human M0 macrophages and highly increased c-jun and NF-κB activation in M1 macrophages. C-jun and p65 are implicated in the effects of cyH on M0 and M1 macrophages since inhibition of their activation prevented the cyH pro-inflammatory effects. In conclusion, we demonstrated that cyH induces or amplifies a pro-inflammatory phenotype in M0 and M1 macrophages by activating JNK/p65 signaling pathway. These results highlight a specific role of cyH in the amplification of tumor-related inflammation by modulating the inflammatory phenotype of macrophages.

High expression levels of egfl7 correlate with low endothelial cell activation in peritumoral vessels of human breast cancer.

Tumor blood vessels participate in the immune response against cancer cells and we previously used pre-clinical models to demonstrate that egfl7 (VE-statin) promotes tumor cell evasion from the immune system by repressing endothelial cell activation, preventing immune cells from entering the tumor mass. In the present study, the expression levels of egfl7 and that of ICAM-1 as a marker of endothelium activation, were evaluated in peritumoral vessels of human breast cancer samples. Breast cancer samples (174 invasive and 30 in situ) from 204 patients treated in 2005 were immunostained for CD31, ICAM-1 and stained for egfl7 using in situ hybridization. The expression levels of ICAM-1 and egfl7 were assessed in peritumoral areas using semi-quantitative scales. There was a strong and significant inverse correlation between the expression of ICAM-1 and that of egfl7 in CD31+ blood vessels. When the ICAM-1 score increased, the egfl7 score reduced significantly (P=0.004), and vice-versa (Cuzick's test for trend across ordered groups). In order to determine which gene influenced the other gene between egfl7 and ICAM-1, the expression levels of either gene were modulated in endothelial cells. Egfl7 regulated ICAM-1 expression while ICAM-1 had no effects on egfl7 expression in the same conditions. Altogether, these results provide further results that egfl7 serves a regulatory role in endothelial cell activation in relation to immune infiltration and that it is a potential therapeutic target to consider for improving anticancer immunotherapies.