Tumor hypoxia modulates podoplanin/CCL21 interactions in CCR7+ NK cell recruitment and CCR7+ tumor cell mobilization.

Podoplanin (PDPN), an O-glycosylated, transmembrane, mucin-type glycoprotein, is expressed by cancer associated fibroblasts (CAFs). In malignant transformation, PDPN is subjected to changes and its role is yet to be established. Here we show that it is involved in modulating the activity of the CCL21/CCR7 chemokine/receptor axis in a hypoxia-dependent manner. In the present model, breast cancer MDA-MB-231 cells and NKL3 cells express the surface CCR7 receptor for CCL21 chemokine which is a potent chemoattractant able to bind to PDPN. The impact of the CCL21/CCR7 axis in the molecular mechanism of the adhesion of NKL3 cells and of MDA-MB-231 breast cancer cells was reduced in a hypoxic tumor environment. In addition to its known effect on migration, CCL21/CCR7 interaction was shown to allow NK cell adhesion to endothelial cells (ECs) and its reduction by hypoxia. A PDPN expressing model of CAFs made it possible to demonstrate the same CCL21/CCR7 axis involvement in the tumor cells to CAFs recognition mechanism through PDPN binding of CCL21. PDPN was induced by hypoxia and its overexpression undergoes a reduction of adhesion, making it an anti-adhesion molecule in the absence of CCL21, in the tumor. CCL21/CCR7 modulated NK cells/ECs and MDA-MB-231 cells/CAF PDPN-dependent interactions were further shown to be linked to hypoxia-dependent microRNAs as miRs: miR-210 and specifically miR-21, miR-29b which influence PDPN expression.

Endothelial precursor cell-based therapy to target the pathologic angiogenesis and compensate tumor hypoxia.

Hypoxia-inducing pathologies as cancer develop pathologic and inefficient angiogenesis which rules tumor facilitating microenvironment, a key target for therapy. As such, the putative ability of endothelial precursor cells (EPCs) to specifically home to hypoxic sites of neovascularization prompted to design optimized, site-specific, cell-mediated, drug-/gene-targeting approach. Thus, EPC lines were established from aorta-gonad-mesonephros (AGM) of murine 10.5 dpc and 11.5 dpc embryo when endothelial repertoire is completed. Lines representing early endothelial differentiation steps were selected: MAgEC10.5 and MagEC11.5. Distinct in maturation, they differently express VEGF receptors, VE-cadherin and chemokine/receptors. MAgEC11.5, more differentiated than MAgEC 10.5, displayed faster angiogenesis in vitro, different response to hypoxia and chemokines. Both MAgEC lines cooperated to tube-like formation with mature endothelial cells and invaded tumor spheroids through a vasculogenesis-like process. In vivo, both MAgEC-formed vessels established blood flow. Intravenously injected, both MAgECs invaded Matrigel(TM)-plugs and targeted tumors. Here we show that EPCs (MAgEC11.5) target tumor angiogenesis and allow local overexpression of hypoxia-driven soluble VEGF-receptor2 enabling drastic tumor growth reduction. We propose that such EPCs, able to target tumor angiogenesis, could act as therapeutic gene vehicles to inhibit tumor growth by vessel normalization resulting from tumor hypoxia alleviation.

Alteration of cathepsin D trafficking induced by hypoxia and extracellular acidification in MCF-7 breast cancer cells.

The microenvironment that surrounds tumor cells is characterized by hypoxic conditions and extracellular acidity. These hostile conditions induce crucial changes in cell behavior and can promote the secretion of many soluble factors such as growth factors, cytokines and enzymes. The lysosomal aspartyl-endopeptidase cathepsin D (CD) is a marker of poor prognosis in breast cancer and is associated with a metastatic risk. In this study, the transport of CD was investigated in a model of breast cancer cells line (MCF-7) cultivated under hypoxia and acidification of media. CD secretion was assessed using Western blot analysis and protease activity was measured in conditioned culture media. We demonstrate that cultured MCF-7 cells secrete an active 52 kDa pCD precursor and report that under hypoxia there was an increased amount of pCD secreted. More surprisingly, extracellular acidification (pH 6 and 5.6) induced the secretion of the fully-mature and active (34 kDa + 14 kDa) double chain CD. Our findings reflect the fact that chemical anomalies influence the secretion path of CD in a breast cancer cell model, resulting in altered trafficking of the mature form. This important result may provide new arguments in favor of the role of extracellular CD in the degradation of the matrix proteins that constitute the breast tumor microenvironment.

Hypoxia-regulated overexpression of soluble VEGFR2 controls angiogenesis and inhibits tumor growth.

VEGFs are found at high levels in hypoxic tumors. As major components directing pathologic neovascularization, they regulate stromal reactions. Consequently, novel strategies targeting and inhibiting VEGF overproduction upon hypoxia offer considerable potential for modern anticancer therapies controlling rather than destroying tumor angiogenesis. Here, we report the design of a vector expressing the soluble form of VEGF receptor-2 (sVEGFR2) driven by a hypoxia-responsive element (HRE)-regulated promoter. To enable in vivo imaging by infrared visualization, mCherry and IFP1.4 coding sequences were built into the vector. Plasmid construction was validated through transfection into embryonic human kidney HEK293 and murine B16F10 melanoma cells. sVEGFR2 was expressed in hypoxic conditions only, confirming that the gene was regulated by the HRE promoter. sVEGFR2 was found to bind efficiently and specifically to murine and human VEGF-A, reducing the growth of tumor and endothelial cells as well as impacting angiogenesis in vitro. The hypoxia-conditioned sVEGFR2 expression was shown to be functional in vivo: Tumor angiogenesis was inhibited and, on stable transfection of B16F10 melanoma cells, tumor growth was reduced. Enhanced expression of sVEGFR2 was accompanied by a modulation in levels of VEGF-A. The resulting balance reflected the effect on tumor growth and on control of angiogenesis. A concomitant increase of intratumor oxygen tension also suggested an influence on vessel normalization. The possibility to express an angiogenesis regulator as sVEGFR2, in a hypoxia-conditioned manner, significantly opens new strategies for tumor vessel-controlled normalization and the design of adjuvants for combined cancer therapies.

Stable tumor vessel normalization with pO2 increase and endothelial PTEN activation by inositol trispyrophosphate brings novel tumor treatment.

Tumor hypoxia is a characteristic of cancer cell growth and invasion, promoting angiogenesis, which facilitates metastasis. Oxygen delivery remains impaired because tumor vessels are anarchic and leaky, contributing to tumor cell dissemination. Counteracting hypoxia by normalizing tumor vessels in order to improve drug and radio therapy efficacy and avoid cancer stem-like cell selection is a highly challenging issue. We show here that inositol trispyrophosphate (ITPP) treatment stably increases oxygen tension and blood flow in melanoma and breast cancer syngeneic models. It suppresses hypoxia-inducible factors (HIFs) and proangiogenic/glycolysis genes and proteins cascade. It selectively activates the tumor suppressor phosphatase and tensin homolog (PTEN) in vitro and in vivo at the endothelial cell (EC) level thus inhibiting PI3K and reducing tumor AKT phosphorylation. These mechanisms normalize tumor vessels by EC reorganization, maturation, pericytes attraction, and lowering progenitor cells recruitment in the tumor. It strongly reduces vascular leakage, tumor growth, drug resistance, and metastasis. ITPP treatment avoids cancer stem-like cell selection, multidrug resistance (MDR) activation and efficiently enhances chemotherapeutic drugs activity. These data show that counteracting tumor hypoxia by stably restoring healthy vasculature is achieved by ITPP treatment, which opens new therapeutic options overcoming hypoxia-related limitations of antiangiogenesis-restricted therapies. By achieving long-term vessels normalization, ITPP should provide the adjuvant treatment required in order to overcome the subtle definition of therapeutic windows for in vivo treatments aimed by the current strategies against angiogenesis-dependent tumors.

News on microenvironmental physioxia to revisit skin cell targeting approaches.

The skin is a multifunctional organ and a first line of defense actively protecting from environmental stress caused by injury, microbial treat, UV irradiation and environmental toxins. Diverse cutaneous cell types together with extracellular matrix elements and factors create a dynamic scene for cellular communication crucial in vital processes such as wound healing, inflammation, angiogenesis, immune response. Direct functional success of skin equilibrium depends on its microenvironment settings and particularly the local oxygen tension. Indeed, skin entire milieu is characterized by and highly dependent on its low oxygen tension called physioxia as emphasized in this review. In the context of skin physioxia, we review and propose here new approaches to minimize age-related changes in skin state and function. We particularly emphasize carbohydrate-mediated interactions and new 3D models of engineered skin substitutes. We highlight newly emerged tools and targets including stem cells, miRNAs, matrix metalloproteinases, mitochondria and natural antioxidants that are promising in prevention of skin ageing and disease restraint. In the era of advanced dermatology, new attempts are bringing us closer to 'well being' perception.

Hypoxia control to normalize pathologic angiogenesis: potential role for endothelial precursor cells and miRNAs regulation.

Tumor microenvironment is a complex and highly dynamic milieu that provides very important clues on tumor development and progression mechanisms. Tumor-associated endothelial cells play a key role in stroma organization. They achieve tumor angiogenesis, a formation of tumor-associated (angiogenic) vessels mainly through sprouting from locally preexisting vessels and/or recruitment of bone marrow-derived endothelial progenitor cells. This process participates to supply nutritional support and oxygen to the growing tumor. Endothelial cells constitute the interface between circulating blood cells, tumor cells and the extracellular matrix, thereby controlling leukocyte recruitment, tumor cell behavior and metastasis formation. Hypoxia, a critical parameter of the tumor microenvironment, controls endothelial/tumor cell interactions and is the key to tumor angiogenesis development. Under hypoxic stress, tumor cells produce factors that promote angiogenesis, vasculogenesis, tumor cell motility, metastasis and cancer stem cell selection. Targeting tumor vessels is a therapeutic strategy that has lately been fast evolving from antiangiogenesis to vessel normalization as discussed in this review. We shall focus on the pivotal role of endothelial cells within the tumor microenvironment, the specific features and the part played by circulating endothelial precursors cells. Attention is stressed on their recruitment to the tumor site and their role in tumor angiogenesis where they are submitted to miRNAs-mediated de/regulation. Here the compensation of the tumor deregulated angiogenic miRNAs - angiomiRs - is emphasized as a potential therapeutic approach. The strategy is to over express anti-angiomiRs in the tumor angiogenesis site upon selective delivery by precursor endothelial cells as miRs carriers.

Myeloid cells are tunable by a polyanionic polysaccharide derivative and co-determine host rescue from lethal virus infection.

Insight into molecular and cellular mechanisms of innate immunity is critical to understand viral pathogenesis and immunopathology and might be exploited for therapy. Whereas the molecular mechanisms of the IFN defense are well established, cellular mechanisms of antiviral immunity are only emerging, and their pharmacological triggering remains unknown. COAM is a polysaccharide derivative with antiviral activity but without comprehension about its mechanism of action. The COAM mixture was fractionated, and prophylactic treatment of mice with COAM polymers of high MW resulted in a conversion from 100% lethal mengovirus infection to an overall survival rate of 93% without obvious clinical sequelae. Differential and quantitative analysis of peritoneal leukocytes demonstrated that COAM induced a profound influx of neutrophils. Selective cell depletion experiments pointed toward neutrophils and macrophages as key effector cells in the rescue of mice from lethal mengovirus. COAM was able to induce mRNA and protein expression of the mouse neutrophil chemokine GCP-2. Binding of GCP-2 to COAM was demonstrated in solution and confirmed by SPR technology. Although COAM was not chemotactic for neutrophils, COAM-anchored muGCP-2 retained chemotactic activity for human and mouse neutrophils. In conclusion, this study established that COAM rescued mice from acute and lethal mengovirus infection by recruiting antiviral leukocytes to the site of infection, as proposed through the induction, binding, and concentration of endogenous chemokines. These findings reinforce the role of neutrophils and macrophages as critical cells that can be manipulated toward antiviral defense.

Carbohydrate-lectin interactions assayed by SPR.

Surface plasmon resonance is a valuable tool to determine the affinity between glycoconjugates and sugar-binding proteins such as plant and animal lectins. The main interest of using such an approach is that neither the lectins - which are proteins - nor their ligands - natural compounds such as glycoproteins, oligosaccharides, polysaccharides, or synthetic glycoconjugates such as glycoclusters or neoglycoproteins - require any tag. Because lectins bear several binding sites, they behave like immunoglobulin eliciting avidity phenomena. This peculiarity may lead to erroneous results if special conditions are not applied. We obtained best and reproducible results when the lectin was immobilized and its ligands were used as soluble analytes. With heterogeneous glycoconjugates such as neoglycoproteins (which are heterogeneous in terms of nature, number, and position of sugar residues) or a mixture of oligosaccharides, the data may be more accurately gathered by using the Sips approach, which has been used to determine mean binding constants of polyclonal antibodies. With small analytes such as oligosaccharides, we found it convenient to determine binding constants by using an inhibitory approach: a neoglycoprotein (M (r) = approximately 80,000) was allowed to bind to the immobilized lectin and small oligosaccharides were used as inhibitors. With larger glycoconjugates such as peptides substituted with glycoclusters, direct binding measurements gave accurate results. Because of the availability of low-cost simple sugars (mono- or disaccharides) it is very convenient to use large concentrations of such carbohydrates to clean the sensor chips instead of more drastic cleaning solutions such as acids or alkali, in such a way that the immobilized lectin is stable for many experiments.

Selective human endothelial cell activation by chemokines as a guide to cell homing.

An original model of organo-specific, immortalized and stabilized endothelial cell lines was used to delineate the part played by some chemokines (CCL21, CX3CL1, CCL5 and CXCL12) and their receptors in endothelium organo-specificity. Chemokine receptor expression and chemokine presentation were investigated on organo-specific human endothelial cell lines. Although the chemokines showed distinct binding patterns for the various endothelial cell lines, these were not correlated with the expression of the corresponding receptors (CX3CR1, CXCR4, CCR5 and CCR7). Experiments with CCL21 on peripheral lymph node endothelial cells demonstrated that the chemokine did not co-localize with its receptor but was associated with extracellular matrix components. The specific activity of chemokines was clearly shown to be related to the endothelial cell origin. Indeed, CX3CL1 and CCL21 promoted lymphocyte recruitment by endothelial cells from the appendix and peripheral lymph nodes, respectively, while CX3CL1 pro-angiogenic activity was restricted to endothelial cells from the appendix and skin. The high specificity of the chemokine/endothelium interaction allowed the design of a direct in vitro endothelial cell targeting assay. This unique cellular model demonstrated a fundamental role for chemokines in conferring on the endothelium its organo-specificity and its potential for tissue targeting through the selective binding, presentation and activation properties of chemokines.

Flow cytometric assay for quantitative and qualitative evaluation of adhesive interactions of tumor cells with endothelial cells.

The purpose of the study was to develop a flow cytometric assay for quantitative determination of adhesive interactions of human endothelial cells (ECs) with tumor cells. EC lines established from human lymph node, appendix, lung, skin and intestine microvessels, labeled with PKH26-GL fluorescent dye, were grown to confluency in 24-well TC plates. Human colon adenocarcinoma cell suspension was overlaid onto labeled ECs, and allowed to adhere for 20 min at 4 degrees C under static conditions. Non-adhering cells were collected first, and adhering tumor cells together with ECs were detached from the culture plate. Collected cell fractions were evaluated by flow cytometry. Results were re-calculated as a ratio (R) of adhering colon carcinoma cells per one EC. We demonstrated that immortalized human microvascular ECs preserved their organ specificity. Colon carcinoma cells adhere preferentially to ECs of intestine origin. The immunofluorescent staining of adhering and non-adhering cancer cell subpopulations has revealed an augmented level of Lewis(x) antigen on adhering cancer cells. The organ specificity of endothelial cell interactions with colon carcinoma cells demonstrated in static conditions was verified and confirmed with flow adhesion assay. The method elaborated is suitable for quantifying of tumor cells adhering to ECs, with simultaneous evaluation of cell surface phenotypic markers of both partner cells participating in adhesive interactions. Validated by comparison to dynamic shear stress adhesion assay in blood flow reconstituted conditions this assay greatly facilitates evaluation of tumor cell-endothelial cell mutual interactions taking place during metastatic process.