Tumour-educated circulating monocytes are powerful candidate biomarkers for diagnosis and disease follow-up of colorectal cancer.

OBJECTIVE: Cancer immunology is a growing field of research whose aim is to develop innovative therapies and diagnostic tests. Starting from the hypothesis that immune cells promptly respond to harmful stimuli, we used peripheral blood monocytes in order to characterise a distinct gene expression profile and to evaluate its potential as a candidate diagnostic biomarker in patients with colorectal cancer (CRC), a still unmet clinical need. DESIGN: We performed a case-control study including 360 peripheral blood monocyte samples from four European oncological centres and defined a gene expression profile specific to CRC. The robustness of the genetic profile and disease specificity were assessed in an independent setting. RESULTS: This screen returned 43 putative diagnostic markers, which we refined and validated in the confirmative multicentric analysis to 23 genes with outstanding diagnostic accuracy (area under the curve (AUC)=0.99 (0.99 to 1.00), Se=100.0% (100.0% to 100.0%), Sp=92.9% (78.6% to 100.0%) in multiple-gene receiver operating characteristic analysis). The diagnostic accuracy was robustly maintained in prospectively collected independent samples (AUC=0.95 (0.85 to 1.00), Se=92.6% (81.5% to 100.0%), Sp=92.3% (76.9% to 100.0%). This monocyte signature was expressed at early disease onset, remained robust over the course of disease progression, and was specific for the monocytic fraction of mononuclear cells. The gene modulation was induced specifically by soluble factors derived from transformed colon epithelium in comparison to normal colon or other cancer histotypes. Moreover, expression changes were plastic and reversible, as they were abrogated upon withdrawal of these tumour-released factors. Consistently, the modified set of genes reverted to normal expression upon curative treatment and was specific for CRC. CONCLUSIONS: Our study is the first to demonstrate monocyte plasticity in response to tumour-released soluble factors. The identified distinct signature in tumour-educated monocytes might be used as a candidate biomarker in CRC diagnosis and harbours the potential for disease follow-up and therapeutic monitoring.

PHD2 regulates arteriogenic macrophages through TIE2 signalling.

Occlusion of the main arterial route redirects blood flow to the collateral circulation. We previously reported that macrophages genetically modified to express low levels of prolyl hydroxylase domain protein 2 (PHD2) display an arteriogenic phenotype, which promotes the formation of collateral vessels and protects the skeletal muscle from ischaemic necrosis. However, the molecular mechanisms underlying this process are unknown. Here, we demonstrate that femoral artery occlusion induces a switch in macrophage phenotype through angiopoietin-1 (ANG1)-mediated Phd2 repression. ANG blockade by a soluble trap prevented the downregulation of Phd2 expression in macrophages and their phenotypic switch, thus inhibiting collateral growth. ANG1-dependent Phd2 repression initiated a feed-forward loop mediated by the induction of the ANG receptor TIE2 in macrophages. Gene silencing and cell depletion strategies demonstrate that TIE2 induction in macrophages is required to promote their proarteriogenic functions, enabling collateral vessel formation following arterial obstruction. These results indicate an indispensable role for TIE2 in sustaining in situ programming of macrophages to a proarteriogenic, M2-like phenotype, suggesting possible new venues for the treatment of ischaemic disorders.

Macrophage skewing by Phd2 haplodeficiency prevents ischaemia by inducing arteriogenesis.

PHD2 serves as an oxygen sensor that rescues blood supply by regulating vessel formation and shape in case of oxygen shortage. However, it is unknown whether PHD2 can influence arteriogenesis. Here we studied the role of PHD2 in collateral artery growth by using hindlimb ischaemia as a model, a process that compensates for the lack of blood flow in case of major arterial occlusion. We show that Phd2 (also known as Egln1) haplodeficient (Phd2(+/-)) mice displayed preformed collateral arteries that preserved limb perfusion and prevented tissue necrosis in ischaemia. Improved arteriogenesis in Phd2(+/-) mice was due to an expansion of tissue-resident, M2-like macrophages and their increased release of arteriogenic factors, leading to enhanced smooth muscle cell (SMC) recruitment and growth. Both chronic and acute deletion of one Phd2 allele in macrophages was sufficient to skew their polarization towards a pro-arteriogenic phenotype. Mechanistically, collateral vessel preconditioning relied on the activation of canonical NF-κB pathway in Phd2(+/-) macrophages. These results unravel how PHD2 regulates arteriogenesis and artery homeostasis by controlling a specific differentiation state in macrophages and suggest new treatment options for ischaemic disorders.

Expression of integrin alpha6beta1 enhances tumorigenesis in glioma cells.

The integrin alpha6beta1 and its main ligand laminin-111 are overexpressed in glioblastoma, as compared with normal brain tissue, suggesting they may be involved in glioblastoma malignancy. To address this question, we stably expressed the alpha6 integrin subunit in the U87 cell line via retroviral-mediated gene transfer. We show that cell surface expression of the alpha6beta1 integrin led to dramatic changes in tumor U87 cell behavior, both in vitro and in vivo. Nude mice receiving either subcutaneous or intracerebral inoculation of alpha6beta1-expressing cells developed substantially more voluminous tumors than mice injected with control cells. The difference in tumor growth was associated with a marked increase in vascularization in response to alpha6beta1 integrin expression and may also be related to changes in the balance between cell proliferation and survival. Indeed, expression of alpha6beta1 enhanced proliferation and decreased apoptosis of U87 cells both in the tumor and in vitro. Additionally, we demonstrate that alpha6beta1 is implicated in glioblastoma cell migration and invasion and that laminin-111 might mediate dissemination of alpha6beta1-positive cells in vivo. Our results highlight for the first time the considerable role of the integrin alpha6beta1 in glioma progression.

alphavbeta5/beta6 integrin suppression leads to a stimulation of alpha2beta1 dependent cell migration resistant to PI3K/Akt inhibition.

Crosstalk between integrins is involved in the regulation of various cell functions including cell migration. Here we identify the interplay between the integrins alphavbeta5/beta6 and alpha2beta1 during cell migration toward type I collagen. Human colon cancer cell lines HT29-D4 and SW480 were used as cell models. To improve our understanding of the consequences of alphavbeta5/beta6 function on alpha2beta1, we decreased the expression of alphav integrins by either siRNA or lysosomal targeting strategies or inhibited their function using, as antagonists, blocking antibodies or disintegrins. In all cases, we observed a greatly enhanced alpha2beta1 integrin-dependent cell migration associated with focal adhesion rearrangements and increased outside-in signaling as demonstrated by elevated phosphorylation of focal adhesion kinase and MAPKinase (ERK1 and ERK2). The alphavbeta5/beta6-dependent limitation of alpha2beta1 function could be overridden by TS2/16, an activating anti-beta1 antibody. Interestingly, compared to control cells, the pharmacological inhibition of PI3Kinase or the siRNA-mediated knockdown of AKT had little effect on the high alpha2beta1-mediated cell migration observed in the absence of alphav integrins or following activation of alpha2beta1 integrins by the TS2/16. These results suggest that integrins alphavbeta5/beta6 repress alpha2beta1 possibly by interfering with their activation process and thereby modify the cell signaling regulation of alpha2beta1-mediated migration.

Early adhesion induces interaction of FAK and Fyn in lipid domains and activates raft-dependent Akt signaling in SW480 colon cancer cells.

Integrin-dependent interaction of epithelial tumor cells with extracellular matrix (ECM) is critical for their migration, but also for hematogenous dissemination. Elevated expression and activity of Src family kinases (SFKs) in colon cancer cells is often required in the disease progression. In this work, we highlighted how focal adhesion kinase (FAK) and SFKs interacted and we analyzed how PI3K/Akt and MAPK/Erk1/2 signaling pathways were activated in early stages of colon cancer cell adhesion. During the first hour, integrin engagement triggered FAK-Y397 phosphorylation and a fraction of FAK was located in lipid rafts/caveolae domains where it interacted with Fyn. The FAK-Y861 and/or -Y925 phosphorylations led to a subsequently FAK translocation out of lipid domains. In parallel, a PI3K/Akt pathway dependent of lipid microdomain integrity was activated. In contrast, the MAPK/Erk1/2 signaling triggered by adhesion increased during at least 4 h and was independent of cholesterol disturbing. Thus, FAK/Fyn interaction in lipid microdomains and a Akt-1 activation occurred at the same time during early contact with ECM suggesting a specific signaling dependent of lipid rafts/caveolae domains.

G alpha(q/11)-coupled P2Y2 nucleotide receptor inhibits human keratinocyte spreading and migration.

Reepithelialization is a critical step in wound healing. It is initiated by keratinocyte migration at the wound edges. After wounding, extracellular nucleotides are released by keratinocytes and other skin cells. Here, we report that activation of P2Y2 nucleotide receptor by ATP/UTP inhibits keratinocyte cell spreading and induces lamellipodium withdrawal. Kymography analysis demonstrates that these effects correlate with a durable decrease of lamellipodium dynamics. P2Y2 receptor activation also induces a dramatic dismantling of the actin network, the loss of alpha3 integrin expression at the cell periphery, and the dissolution of focal contacts as indicated by the alteration of alpha(v) integrins and focal contact protein distribution. In addition, activation of P2Y2R prevents growth factor-induced phosphorylation of Erk(1,2) and Akt/PkB. The use of a specific pharmacological inhibitor (YM-254890), the depletion of G alpha(q/11) by siRNA, or the expression of a constitutively active G alpha(q/11) mutant (Q209L) show that activation of G alpha(q/11) is responsible for these ATP/UTP-induced effects. Finally, we report that ATP delays growth factor-induced wound healing of keratinocyte monolayers. Collectively, these findings provide evidence for a unique and important role for extracellular nucleotides as efficient autocrine/paracrine regulators of keratinocyte shape and migration during wound healing.

Lebectin and lebecetin, two C-type lectins from snake venom, inhibit alpha5beta1 and alphaV-containing integrins.

Integrins are essential protagonists in the complex multistep process of cancer progression and metastasis. We recently reported that lebectin, a novel C-type lectin from Macrovipera lebetina venom, displays an anti-integrin activity. In this study, we extend this observation to lebecetin, a second C-type lectin isolated from the same venom and previously reported as a potent inhibitor of platelet aggregation. Both venom lectins appear to exert their effect on cell adhesion, migration, invasion and proliferation by inhibiting alpha5beta1 and alphav-containing integrins. Moreover, the inhibition of alpha5beta1 and alphav integrins is likely due to the binding of venom peptides, as both lebectin and lebecetin co-immunoprecipitate with these integrins. Lebectin and lebecetin are thus the first examples of venom C-type lectins inhibiting an integrin other than the collagen receptor alpha2beta1.