Marcin Wysoczynski

CXCR4-SDF-1 signalling, locomotion, chemotaxis and adhesion.

Chemokines, small pro-inflammatory chemoattractant cytokines, that bind to specific G-protein-coupled seven-span transmembrane receptors present on plasma membranes of target cells are the major regulators of cell trafficking. In addition some chemokines have been reported to modulate cell survival and growth. Moreover, compelling evidence is accumulating that cancer cells may employ several mechanisms involving chemokine–chemokine receptor axes during their metastasis that also regulate the trafficking of normal cells. Of all the chemokines, stromal-derived factor-1 (SDF-1), an α-chemokine that binds to G-protein-coupled CXCR4, plays an important and unique role in the regulation of stem/progenitor cell trafficking. First, SDF-1 regulates the trafficking of CXCR4+ haemato/lymphopoietic cells, their homing/retention in major haemato/lymphopoietic organs and accumulation of CXCR4+ immune cells in tissues affected by inflammation. Second, CXCR4 plays an essential role in the trafficking of other tissue/organ specific stem/progenitor cells expressing CXCR4 on their surface, e.g., during embryo/organogenesis and tissue/organ regeneration. Third, since CXCR4 is expressed on several tumour cells, these CXCR4 positive tumour cells may metastasize to the organs that secrete/express SDF-1 (e.g., bones, lymph nodes, lung and liver). SDF-1 exerts pleiotropic effects regulating processes essential to tumour metastasis such as locomotion of malignant cells, their chemoattraction and adhesion, as well as plays an important role in tumour vascularization. This implies that new therapeutic strategies aimed at blocking the SDF-1–CXCR4 axis could have important applications in the clinic by modulating the trafficking of haemato/lymphopoietic cells and inhibiting the metastatic behaviour of tumour cells as well. In this review, we focus on a role of the SDF-1–CXCR4 axis in regulating the metastatic behaviour of tumour cells and discuss the molecular mechanisms that are essential to this process.

Migration of Bone Marrow and Cord Blood Mesenchymal Stem Cells In Vitro Is Regulated by Stromal‐Derived Factor‐1‐CXCR4 and Hepatocyte Growth Factor‐c‐met Axes and Involves Matrix Metalloproteinases

Human mesenchymal stem cells (MSCs) are increasingly being considered in cell‐based therapeutic strategies for regeneration of various organs/tissues. However, the signals required for their homing and recruitment to injured sites are not yet fully understood. Because stromal‐derived factor (SDF)‐1 and hepatocyte growth factor (HGF) become up‐regulated during tissue/organ damage, in this study we examined whether these factors chemoattract ex vivo‐expanded MSCs derived from bone marrow (BM) and umbilical cord blood (CB). Specifically, we investigated the expression by MSCs of CXCR4 and c‐met, the cognate receptors of SDF‐1 and HGF, and their functionality after early and late passages of MSCs. We also determined whether MSCs express matrix metalloproteinases (MMPs), including membrane type 1 (MT1)‐MMP, matrix‐degrading enzymes that facilitate the trafficking of hematopoietic stem cells. We maintained expanded BM‐ or CB‐derived MSCs for up to 15–18 passages with monitoring of the expression of 1) various tissue markers (cardiac and skeletal muscle, neural, liver, and endothelial cells), 2) functional CXCR4 and c‐met, and 3) MMPs. We found that for up to 15–18 passages, both BM‐ and CB‐derived MSCs 1) express mRNA for cardiac, muscle, neural, and liver markers, as well as the vascular endothelial (VE) marker VE‐cadherin; 2) express CXCR4 and c‐met receptors and are strongly attracted by SDF‐1 and HGF gradients; 3) express MMP‐2 and MT1‐MMP transcripts and proteins; and 4) are chemo‐invasive across the reconstituted basement membrane Matrigel. These in vitro results suggest that the SDF‐1‐CXCR4 and HGF‐c‐met axes, along with MMPs, may be involved in recruitment of expanded MSCs to damaged tissues.

Microvesicles derived from activated platelets induce metastasis and angiogenesis in lung cancer.

The role of platelets in tumor progression and metastasis has been recognized but the mechanism of their action remains unclear. Five human lung cancer cell lines (A549, CRL 2066, CRL 2062, HTB 183, HTB 177) and a murine Lewis lung carcinoma (LCC) cell line (for an in vivo model of metastasis) were used to investigate how platelet‐derived microvesicles (PMV), which are circular fragments shed from the surface membranes of activated platelets, and exosomes released from platelet α‐granules, could contribute to metastatic spread. We found that PMV transferred the platelet‐derived integrin CD41 to most of the lung cancer cell lines tested and stimulated the phosphorylation of mitogen‐activated protein kinase p42/44 and serine/threonine kinase as well as the expression of membrane type 1‐matrix metalloproteinase (MT1‐MMP). PMV chemoattracted 4 of the 5 cell lines, with the highly metastatic A549 cells exhibiting the strongest response. In A549 cells, PMV were shown to stimulate proliferation, upregulate cyclin D2 expression and increase trans‐Matrigel chemoinvasion. Furthermore, in these cells, PMV stimulated mRNA expression for angiogenic factors such as MMP‐9, vascular endothelial growth factor, interleukin‐8 and hepatocyte growth factor, as well as adhesion to fibrinogen and human umbilical vein endothelial cells. Intravenous injection of murine PMV‐covered LLC cells into syngeneic mice resulted in significantly more metastatic foci in their lungs and LLC cells in bone marrow than in control animals injected with LCC cells not covered with PMV. Based on these findings, we suggest that PMV play an important role in tumor progression/metastasis and angiogenesis in lung cancer.

Cells Expressing Early Cardiac Markers Reside in the Bone Marrow and Are Mobilized Into the Peripheral Blood After Myocardial Infarction

The concept that bone marrow (BM)–derived cells participate in cardiac regeneration remains highly controversial and the identity of the specific cell type(s) involved remains unknown. In this study, we report that the postnatal BM contains a mobile pool of cells that express early cardiac lineage markers (Nkx2.5/Csx, GATA-4, and MEF2C). These cells are present in significant amounts in BM harvested from young mice but their abundance decreases with age; in addition, the responsiveness of these cells to gradients of motomorphogens SDF-1, HGF, and LIF changes with age. FACS analysis, combined with analysis of early cardiac markers at the mRNA and protein levels, revealed that cells expressing these markers reside in the nonadherent, nonhematopoietic CXCR4+/Sca-1+/lin−/CD45− mononuclear cell (MNC) fraction in mice and in the CXCR4+/CD34+/AC133+/CD45− BMMNC fraction in humans. These cells are mobilized into the peripheral blood after myocardial infarction and chemoattracted to the infarcted myocardium in an SDF-1-CXCR4–, HGF-c-Met–, and LIF-LIF-R–dependent manner. To our knowledge, this is the first demonstration that the postnatal BM harbors a nonhematopoietic population of cells that express markers for cardiac differentiation. We propose that these potential cardiac progenitors may account for the myocardial regenerative effects of BM. The present findings provide a novel paradigm that could reconcile current controversies and a rationale for investigating the use of BM-derived cardiac progenitors for myocardial regeneration.

Novel insight into stem cell mobilization-plasma sphingosine-1-phosphate is a major chemoattractant that directs the egress of hematopoietic stem progenitor cells from the bone marrow and its level in peripheral blood increases during mobilization due to activation of complement cascade/membrane attack complex.

The complement cascade (CC) becomes activated and its cleavage fragments play a crucial role in the mobilization of hematopoietic stem/progenitor cells (HSPCs). Here, we sought to determine which major chemoattractant present in peripheral blood (PB) is responsible for the egress of HSPCs from the bone marrow (BM). We noticed that normal and mobilized plasma strongly chemoattracts HSPCs in a stromal-derived factor-1 (SDF-1)-independent manner because (i) plasma SDF-1 level does not correlate with mobilization efficiency; (ii) the chemotactic plasma gradient is not affected in the presence of AMD3100 and (iii) it is resistant to denaturation by heat. Surprisingly, the observed loss of plasma chemotactic activity after charcoal stripping suggested the involvement of bioactive lipids and we focused on sphingosine-1-phosphate (S1P), a known chemoattracant of HSPCs. We found that S1P (i) creates in plasma a continuously present gradient for BM-residing HSPCs; (ii) is at physiologically relevant concentrations a chemoattractant several magnitudes stronger than SDF-1 and (iii) its plasma level increases during mobilization due to CC activation and interaction of the membrane attack complex (MAC) with erythrocytes that are a major reservoir of S1P. We conclude and propose a new paradigm that S1P is a crucial chemoattractant for BM-residing HSPCs and that CC through MAC induces the release of S1P from erythrocytes for optimal egress/mobilization of HSPCs.

Lung cancer secreted microvesicles: Underappreciated modulators of microenvironment in expanding tumors

Microvesicles (MVs) are shed from cell membranes of several cell types and have an important function in cell‐to‐cell communication. Exponentially growing lung cancer cells secrete large quantities of MVs and we were interested in their role in tumor progression. We observed that both human and murine lung cancer cell lines secrete more MVs in response to non‐apoptotic doses of hypoxia and irradiation. These tumor‐derived (t)MVs activate and chemoattract stroma fibroblasts and endothelial cells. Furthermore, they induce expression of several pro‐angiopoietic factors in stromal cells such as IL‐8, VEGF, LIF, OSM, IL‐11 and MMP‐9. We also noticed that conditioned media harvested from stroma cells stimulated by tMVs enhanced the metastatic potential of both human and murine lung cancer cells in vivo. Thus, we postulated that tMVs are underappreciated constituents of the tumor microenvironment and play a pivotal role in tumor progression, metastasis and angiogenesis.

Novel epigenetic mechanisms that control pluripotency and quiescence of adult bone marrow-derived Oct4 + very small embryonic-like stem cells

Recently, we identified in adult tissues a population of Oct4+SSEA-1+Sca-1+lin−CD45− very small embryonic-like stem cells (VSELs). First, to address recent controversies on Oct4 expression in cells isolated from adult organs, we show here evidence that Oct4 promoter in bone marrow (BM)-derived VSELs has an open chromatin structure and is actively transcribed. Next, to explain VSELs quiescence and lack of teratoma formation, we demonstrate a unique DNA methylation pattern at some developmentally crucial imprinted genes, showing hypomethylation/erasure of imprints in paternally methylated and hypermethylation of imprints in maternally methylated ones. These epigenetic characteristics leading to upregulation in VSELs of H19 and p57KIP2 (also known as Cdkn1c) and repression of Igf2 and Rasgrf1 explain VSELs quiescent status. Interestingly, this unique pattern in imprinted gene methylation is reverted in cocultures with a C2C12 supportive cell-line when VSELs are induced to form VSEL-derived spheres (VSEL-DSs) enriched for stem cells able to differentiate into all three germ layers. Therefore, we suggest that the proliferative/developmental potential of Oct4+ VSELs is epigenetically regulated by expression of Oct4 and some imprinted genes, and postulate that restoring the proper methylation pattern of imprinted genes will be a crucial step for using these cells in regenerative medicine.

Enhancing effect of platelet‐derived microvesicles on the invasive potential of breast cancer cells

BACKGROUND:  Platelets (PLTs) have been postulated to play a role in cancer progression and metastasis. Recently, it was demonstrated that PLT‐derived microvesicles (PMVs) transfer various surface receptors and/or adhesion molecules to target cells and modulate their biological responses. In this work, it was hypothesized that PMVs interact with breast cancer cells, increasing their invasiveness.

Evidence That Very Small Embryonic‐Like Stem Cells Are Mobilized into Peripheral Blood

Recently, we identified in murine adult tissues, including bone marrow, a population of very small embryonic‐like (VSEL) stem cells. Here, we provide further evidence that under steady‐state conditions these cells circulate at very low levels in peripheral blood (PB) (∼100–200 cells/ml) and could be additionally mobilized during pharmacological granulocyte‐colony‐stimulating factor‐induced or stress‐related mobilization, as demonstrated in a model of toxic liver or skeletal muscle damage induced by injection of carbon tetrachloride or cardiotoxin, respectively. The number of circulating VSEL stem cells under steady‐state conditions in PB of 2‐month‐old animals was five times higher than that in 1‐year‐old mice. In conclusion, this study supports a hypothesis that VSEL stem cells are a mobile pool of primitive stem cells that could be released from the stem cell niches into PB. Further studies are needed, however, to see whether the level of these cells circulating in PB could become a prognostic indicator to assess the regenerative potential of an adult organism and/or clinical outcome from an injury.

The role of stromal-derived factor-1--CXCR7 axis in development and cancer.

Cancer metastasis is a major clinical problem that contributes to unsuccessful therapy. Augmenting evidence indicates that metastasizing cancer cells employ several mechanisms that are involved in developmental trafficking of normal stem cells. Stromal-derived factor-1 (SDF-1) is an important alpha-chemokine that binds to the G-protein-coupled seven-transmembrane span CXCR4. The SDF-1-CXCR4 axis regulates trafficking of normal and malignant cells. SDF-1 is an important chemoattractant for a variety of cells including hematopoietic stem/progenitor cells. For many years, it was believed that CXCR4 was the only receptor for SDF-1. However, several reports recently provided evidence that SDF-1 also binds to another seven-transmembrane span receptor called CXCR7, sharing this receptor with another chemokine family member called Interferon-inducible T-cell chemoattractant (I-TAC). Thus, with CXCR7 identified as a new receptor for SDF-1, the role of the SDF-1-CXCR4 axis in regulating several biological processes becomes more complex. Based on the available literature, this review addresses the biological significance of SDF-1s interaction with CXCR7, which may act as a kind of decoy or signaling receptor depending on cell type. Augmenting evidence suggests that CXCR7 is involved in several aspects of tumorogenesis and could become an important target for new anti-metastatic and anti-cancer drugs.