The prospect of tumor microenvironment-modulating therapeutical strategies.

Multiple mechanisms promote tumor prosperity, which does not only depend on cell-autonomous, inherent abnormal characteristics of the malignant cells that facilitate rapid cell division and tumor expansion. The neoplastic tissue is embedded in a supportive and dynamic tumor microenvironment (TME) that nurtures and protects the malignant cells, maintaining and perpetuating malignant cell expansion. The TME consists of different elements, such as atypical vasculature, various innate and adaptive immune cells with immunosuppressive or pro-inflammatory properties, altered extracellular matrix (ECM), activated stromal cells, and a wide range of secreted/stroma-tethered bioactive molecules that contribute to malignancy, directly or indirectly. In this review, we describe the various TME components and provide examples of anti-cancer therapies and novel drugs under development that aim to target these components rather than the intrinsic processes within the malignant cells. Combinatory TME-modulating therapeutic strategies may be required to overcome the resistance to current treatment options and prevent tumor recurrence.

Estrogen receptor inhibition enhances cold-induced adipocyte beiging and glucose tolerance.

BACKGROUND:  Low estrogen states, exemplified by postmenopausal women, are associated with increased adiposity and metabolic dysfunction. We recently reported a paradox, in which a conditional estrogen receptor-alpha (ERα) mutant mouse shows a hyper-metabolic phenotype with enhanced brown/beige cell formation ("browning/beiging"). HYPOTHESIS:  These observations led us to consider that although systemic deficiency of estrogen or ERα in mice results in obesity and glucose intolerance at room temperature, cold exposure might induce enhanced browning/beiging and improve glucose metabolism. METHODS AND RESULTS:  Remarkably, studying cold-exposure in mouse models of inhibited estrogen signaling - ERαKO mice, ovariectomy, and treatment with the ERα antagonist Fulvestrant - supported this notion. ERα/estrogen-deficient mice demonstrated enhanced cold-induced beiging, reduced adiposity and improved glucose tolerance. Fulvestrant was also effective in diet-induced obesity settings. Mechanistically, ERα inhibition sensitized cell-autonomous beige cell differentiation and stimulation, including ß3-adrenoreceptor-dependent adipocyte beiging. CONCLUSION:  Taken together, our findings highlight a therapeutic potential for obese/diabetic postmenopausal patients; cold exposure is therefore predicted to metabolically benefit those patients.

Form(ul)ation of adipocytes by lipids.

Lipids have the potential to serve as bio-markers, which allow us to analyze and to identify cells under various experimental settings, and to serve as a clinical diagnostic tool. For example, diagnosis according to specific lipids that are associated with diabetes and obesity. The rapid development of mass-spectrometry techniques enables identification and profiling of multiple types of lipid species. Together, lipid profiling and data interpretation forge the new field of lipidomics. Lipidomics can be used to characterize physiologic and pathophysiological processes in adipocytes, since lipid metabolism is at the core of adipocyte physiology and energy homeostasis. A significant bulk of lipids are stored in adipocytes, which can be released and used to produce energy, used to build membranes, or used as signaling molecules that regulate metabolism. In this review, we discuss how exhaust of lipidomes can be used to study adipocyte differentiation, physiology and pathophysiology.

Oestrogen signalling in white adipose progenitor cells inhibits differentiation into brown adipose and smooth muscle cells.

Oestrogen, often via oestrogen receptor alpha (ERα) signalling, regulates metabolic physiology, highlighted by post-menopausal temperature dysregulation (hot flashes), glucose intolerance, increased appetite and reduced metabolic rate. Here we show that ERα signalling has a role in adipose lineage specification in mice. ERα regulates adipose progenitor identity and potency, promoting white adipogenic lineage commitment. White adipose progenitors lacking ERα reprogramme and enter into smooth muscle and brown adipogenic fates. Mechanistic studies highlight a TGFß programme involved in progenitor reprogramming downstream of ERα signalling. The observed reprogramming has profound metabolic outcomes; both female and male adipose-lineage ERα-mutant mice are lean, have improved glucose sensitivity and are resistant to weight gain on a high-fat diet. Further, they are hypermetabolic, hyperphagic and hyperthermic, all consistent with a brown phenotype. Together, these findings indicate that ERα cell autonomously regulates adipose lineage commitment, brown fat and smooth muscle cell formation, and systemic metabolism, in a manner relevant to prevalent metabolic diseases.

GSK3ß regulates physiological migration of stem/progenitor cells via cytoskeletal rearrangement.

Regulation of hematopoietic stem and progenitor cell (HSPC) steady-state egress from the bone marrow (BM) to the circulation is poorly understood. While glycogen synthase kinase-3ß (GSK3ß) is known to participate in HSPC proliferation, we revealed an unexpected role in the preferential regulation of CXCL12-induced migration and steady-state egress of murine HSPCs, including long-term repopulating HSCs, over mature leukocytes. HSPC egress, regulated by circadian rhythms of CXCL12 and CXCR4 levels, correlated with dynamic expression of GSK3ß in the BM. Nevertheless, GSK3ß signaling was CXCL12/CXCR4 independent, suggesting that synchronization of both pathways is required for HSPC motility. Chemotaxis of HSPCs expressing higher levels of GSK3ß compared with mature cells was selectively enhanced by stem cell factor-induced activation of GSK3ß. Moreover, HSPC motility was regulated by norepinephrine and insulin-like growth factor-1 (IGF-1), which increased or reduced, respectively, GSK3ß expression in BM HSPCs and their subsequent egress. Mechanistically, GSK3ß signaling promoted preferential HSPC migration by regulating actin rearrangement and microtubuli turnover, including CXCL12-induced actin polarization and polymerization. Our study identifies a previously unknown role for GSK3ß in physiological HSPC motility, dictating an active, rather than a passive, nature for homeostatic egress from the BM reservoir to the blood circulation.

Quantifying hematopoietic stem and progenitor cell mobilization.

Allogeneic donor blood cells and autologous peripheral blood leukocytes (PBL), obtained following -clinical mobilization procedures, are routinely used as a major source of hematopoietic stem and progenitor cells (HSPC) for transplantation protocols. It is, therefore, essential to evaluate and to quantify the extent by which the HSPC are mobilized and enriched in the circulation in correlation with their long-term hematopoietic reconstitution capacity. In this chapter, we describe quantitative methods that measure the number of mobilized HSPC according to specific criteria, as well as their functional properties in vitro and in vivo. The described assays are useful for assessment of progenitor cell mobilization as applied to both human and murine HSPC.

S1P promotes murine progenitor cell egress and mobilization via S1P1-mediated ROS signaling and SDF-1 release.

The mechanisms of hematopoietic progenitor cell egress and clinical mobilization are not fully understood. Herein, we report that in vivo desensitization of Sphingosine-1-phosphate (S1P) receptors by FTY720 as well as disruption of S1P gradient toward the blood, reduced steady state egress of immature progenitors and primitive Sca-1(+)/c-Kit(+)/Lin(-) (SKL) cells via inhibition of SDF-1 release. Administration of AMD3100 or G-CSF to mice with deficiencies in either S1P production or its receptor S1P(1), or pretreated with FTY720, also resulted in reduced stem and progenitor cell mobilization. Mice injected with AMD3100 or G-CSF demonstrated transient increased S1P levels in the blood mediated via mTOR signaling, as well as an elevated rate of immature c-Kit(+)/Lin(-) cells expressing surface S1P(1) in the bone marrow (BM). Importantly, we found that S1P induced SDF-1 secretion from BM stromal cells including Nestin(+) mesenchymal stem cells via reactive oxygen species (ROS) signaling. Moreover, elevated ROS production by hematopoietic progenitor cells is also regulated by S1P. Our findings reveal that the S1P/S1P(1) axis regulates progenitor cell egress and mobilization via activation of ROS signaling on both hematopoietic progenitors and BM stromal cells, and SDF-1 release. The dynamic cross-talk between S1P and SDF-1 integrates BM stromal cells and hematopoeitic progenitor cell motility.

CD45 regulates homing and engraftment of immature normal and leukemic human cells in transplanted immunodeficient mice.

Bone marrow homing and engraftment by clinically transplanted hematopoietic stem and progenitor cells is a complex process that is not fully understood. We report that the pan-leukocyte CD45 phosphatase plays an essential role in trafficking and repopulation of the bone marrow by immature human CD34(+) cells and leukemic cells in transplanted nonobese diabetic severe combined immunodeficient mice. Inhibiting CD45 function by blocking antibodies or a CD45 inhibitor impaired the motility of both normal and leukemic human cells. Blocking CD45 inhibited homing and repopulation by immature human CD34(+) cells as well as homing of primary patient leukemic cells. In addition, CD45 inhibition negatively affected development of hematopoietic progenitors in vitro and their recovery in transplanted recipients in vivo, revealing the central role of CD45 in the regulation of hematopoiesis. Moreover, CD45 blockage induced a hyperadhesive phenotype in immature human progenitor cells as well as in murine leukocytes, leading to their defective adhesion interactions with endothelial cells. This phenotype was further manifested by the ability of CD45 blockage to prevent breakdown of adhesion interactions in the BM, which inhibited murine progenitor mobilization. The substantial effects of a direct CD45 inhibition point at its essential roles in cell trafficking, including murine progenitor cell mobilization and both normal immature and leukemic human hematopoietic cells as well as regulation of hematopoiesis and engraftment potential.

Pathways implicated in stem cell migration: the SDF-1/CXCR4 axis.

The hallmark of hematopoietic stem and progenitor cells (HSPCs) is their motility, which is essential for their function, such as recruitment upon demand. Stromal Derived Factor-1 (SDF-1, CXCL12) and its major receptor CXCR4 play major roles in stem cell motility and development. In vitro migration assays, implicating either gradients or cell surface-bound forms of SDF-1, are easy to perform and provide vital information regarding directional and random stem cell motility, which correlate with their repopulation potential in clinical and experimental transplantations. In vivo stem cell homing to the bone marrow, their retention, engraftment, and egress to the circulation, all involve SDF-1/CXCR4 interactions. Finally, other stem cell features such as stem cell survival and proliferation, are also dependent on the SDF-1/CXCR4 axis.

Rapid mobilization of hematopoietic progenitors by AMD3100 and catecholamines is mediated by CXCR4-dependent SDF-1 release from bone marrow stromal cells.

Steady-state egress of hematopoietic progenitor cells can be rapidly amplified by mobilizing agents such as AMD3100, the mechanism, however, is poorly understood. We report that AMD3100 increased the homeostatic release of the chemokine stromal cell derived factor-1 (SDF-1) to the circulation in mice and non-human primates. Neutralizing antibodies against CXCR4 or SDF-1 inhibited both steady state and AMD3100-induced SDF-1 release and reduced egress of murine progenitor cells over mature leukocytes. Intra-bone injection of biotinylated SDF-1 also enhanced release of this chemokine and murine progenitor cell mobilization. AMD3100 directly induced SDF-1 release from CXCR4(+) human bone marrow osteoblasts and endothelial cells and activated uPA in a CXCR4/JNK-dependent manner. Additionally, ROS inhibition reduced AMD3100-induced SDF-1 release, activation of circulating uPA and mobilization of progenitor cells. Norepinephrine treatment, mimicking acute stress, rapidly increased SDF-1 release and progenitor cell mobilization, whereas ß2-adrenergic antagonist inhibited both steady state and AMD3100-induced SDF-1 release and progenitor cell mobilization in mice. In conclusion, this study reveals that SDF-1 release from bone marrow stromal cells to the circulation emerges as a pivotal mechanism essential for steady-state egress and rapid mobilization of hematopoietic progenitor cells, but not mature leukocytes.

CD45 regulates retention, motility, and numbers of hematopoietic progenitors, and affects osteoclast remodeling of metaphyseal trabecules.

The CD45 phosphatase is uniquely expressed by all leukocytes, but its role in regulating hematopoietic progenitors is poorly understood. We show that enhanced CD45 expression on bone marrow (BM) leukocytes correlates with increased cell motility in response to stress signals. Moreover, immature CD45 knockout (KO) cells showed defective motility, including reduced homing (both steady state and in response to stromal-derived factor 1) and reduced granulocyte colony-stimulating factor mobilization. These defects were associated with increased cell adhesion mediated by reduced matrix metalloproteinase 9 secretion and imbalanced Src kinase activity. Poor mobilization of CD45KO progenitors by the receptor activator of nuclear factor kappaB ligand, and impaired modulation of the endosteal components osteopontin and stem cell factor, suggested defective osteoclast function. Indeed, CD45KO osteoclasts exhibited impaired bone remodeling and abnormal morphology, which we attributed to defective cell fusion and Src function. This led to irregular distribution of metaphyseal bone trabecules, a region enriched with stem cell niches. Consequently, CD45KO mice had less primitive cells in the BM and increased numbers of these cells in the spleen, yet with reduced homing and repopulation potential. Uncoupling environmental and intrinsic defects in chimeric mice, we demonstrated that CD45 regulates progenitor movement and retention by influencing both the hematopoietic and nonhematopoietic compartments.

Use of lipophilic near-infrared dye in whole-body optical imaging of hematopoietic cell homing.

We develop an optical whole-body imaging technique for monitoring normal and leukemic hematopoietic cell homing in vivo. A recently developed near-infrared (NIR) lipophilic carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR) is used to safely and directly label the membranes of human leukemic Pre-B ALL G2 cell lines as well as primary murine lymphocytes and erythrocytes. DiR has absorption and fluorescence maxima at 750 and 782 nm, respectively, which corresponds to low light absorption and autofluorescence in living tissues. This allows us to obtain a significant signal with very low background level. A charge-coupled device (CCD)-based imager is used for noninvasive whole-body imaging of DiR-labeled cell homing in intact animals. This powerful technique can potentially visualize any cell type without use of specific antibodies conjugated with NIR fluorescent tag or loading cells with transporter-delivered NIR fluorophores. Thus, in vivo imaging based on NIR lipophilic carbocyanine dyes in combination with advanced optical techniques may serve as a powerful alternative or complementation to other small animal imaging methods.

Osteoclasts degrade endosteal components and promote mobilization of hematopoietic progenitor cells.

Here we investigated the potential role of bone-resorbing osteoclasts in homeostasis and stress-induced mobilization of hematopoietic progenitors. Different stress situations induced activity of osteoclasts (OCLs) along the stem cell-rich endosteum region of bone, secretion of proteolytic enzymes and mobilization of progenitors. Specific stimulation of OCLs with RANKL recruited mainly immature progenitors to the circulation in a CXCR4- and MMP-9-dependent manner; however, RANKL did not induce mobilization in young female PTPepsilon-knockout mice with defective OCL bone adhesion and resorption. Inhibition of OCLs with calcitonin reduced progenitor egress in homeostasis, G-CSF mobilization and stress situations. RANKL-stimulated bone-resorbing OCLs also reduced the stem cell niche components SDF-1, stem cell factor (SCF) and osteopontin along the endosteum, which was associated with progenitor mobilization. Finally, the major bone-resorbing proteinase, cathepsin K, also cleaved SDF-1 and SCF. Our findings indicate involvement of OCLs in selective progenitor recruitment as part of homeostasis and host defense, linking bone remodeling with regulation of hematopoiesis.

Meet the multifunctional and sexy glycoforms of glycodelin.

Glycodelin, a human-secreted glycoprotein that appears in a small number of glycoforms, exhibits diverse biological activities, such as in contraception and immunosuppression. Moreover, different tissue-specific glycoforms appear to mediate diverse functions. Quite unusually, the glycodelin N-linked glycans differ between the male and female glycoforms. The fact that these glycans are fundamental for exerting the physiological activities of the different glycoforms, makes them an interesting target for glycobiology research. This review will focus on the involvement of the glycans in glycodelin activity and compare between the several glycoforms.