Endothelial Progenitor Research Articles

Concise Review: Stem Cells for Corneal Wound Healing.

Corneal wound healing is a complex process that occurs in response to various injuries and commonly used refractive surgery. It is a significant clinical problem, which may lead to serious complications due to either incomplete (epithelial) or excessive (stromal) healing. Epithelial stem cells clearly play a role in this process, whereas the contribution of stromal and endothelial progenitors is less well studied. The available evidence on stem cell participation in corneal wound healing is reviewed, together with the data on the use of corneal and non‐corneal stem cells to facilitate this process in diseased or postsurgical conditions. Important aspects of corneal stem cell generation from alternative cell sources, including pluripotent stem cells, for possible transplantation upon corneal injuries or in disease conditions are also presented. Stem Cells 2017;35:2105–2114

Endothelial colony-forming cells and pro-angiogenic cells: clarifying definitions and their potential role in mitigating acute kidney injury.

Acute kidney injury (AKI) represents a significant clinical concern that is associated with high mortality rates and also represents a significant risk factor for the development of chronic kidney disease (CKD). This article will consider alterations in renal endothelial function in the setting of AKI that may underlie impairment in renal perfusion and how inefficient vascular repair may manifest post-AKI and contribute to the potential transition to CKD. We provide updated terminology for cells previously classified as 'endothelial progenitor' that may mediate vascular repair such as pro-angiogenic cells and endothelial colony-forming cells. We consider how endothelial repair may be mediated by these different cell types following vascular injury, particularly in models of AKI. We further summarize the potential ability of these different cells to mitigate the severity of AKI, improve perfusion and maintain vascular structure in pre-clinical studies.

Abstract 794: Potential role of DLL4 in uveal melanoma vascular mimicry

Abstract Uveal melanoma is the most common malignancy of the eye. Thanks to gene array analysis it is possible to classify uveal melanoma in Class 1 (low metastasis risk) and Class 2 (high metastasis risk) tumor. This classification will ultimately determine the tumor treatment, risk of metastasis and patient surveillance. Progression to metastasis remains by far the greatest problem in uveal melanoma and is associated with loss of BAP1 tumor suppressor. Bioinformatic analyses of RNA-Seq indicated that pro-angiogenic genes such as DLL4, VEGFA, VEGFC and HIF1a are overexpressed in Class 2 compared to Class 1 uveal melanoma while angiogenic inhibitors such as ZFP36L1, HIF1AN, VEGFB, VHL and HIF3A are downregulated. Further, we found that DLL4 is among the 5 most highly overexpressed genes associated with BAP1 loss in clinical specimens and in uveal melanoma cell lines induced to deplete BAP1. DLL4 is a Notch ligand known to regulate endothelial cells, bone marrow endothelial cell progenitors and angiogenesis. We hypothesize that DLL4 contributes to vascular mimicry in uveal melanoma. To test this hypothesis, we will test uveal melanoma cell lines induced to deplete BAP1 using shRNA in cell culture-based and in vivo models. The results of this research have the potential to elucidate the mechanism by which vascular mimicry occurs in uveal melanoma. Citation Format: Julia Escandon, Matthew G. Field, Stefan Kurtenbach, Jeffim Kuznetzov, Christina L. Decatur, J William Harbour. Potential role of DLL4 in uveal melanoma vascular mimicry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 794. doi:10.1158/1538-7445.AM2017-794

Vascular Regenerative Surgery: Promised Land for Tissue Engineers?

Cardiovascular cell therapy is a promising new field for the development of treatments for cardiovascular diseases, which remain a major cause of mortality around the world. In this review, we highlight the options currently available for the development of specific cell therapy approaches applied to regeneration of cardiac and vascular tissues. Different cell types have attracted a lot of attention and extensive investigations for the treatment of vascular diseases, including embryonic stem cells, mesenchymal stem cells, induced pluripotent stem cells, and endothelial progenitors. The combination of human cells and increasingly safe and physiologically compliant biomaterials is currently offering an unprecedented opportunity to develop effective cell therapy for either major blood vessels or the microvasculature. Efficacy and safety of cell therapy are the challenges for the new generation of regenerative medicine scientists determined to develop new remedies for cardiovascular diseases. Here we present the state of the art in this biomedical field and the options in terms of cell types and biomaterials currently available for cardiovascular cell therapy.

DMSO Promotes Hematopoietic Differentiation of Human Embryonic Stem Cells in vitro

To explore the role of dimethyl sulfoxide (DMSO) in the hematopoietic differentiation of human embryonic stem cells (hESCs). The role of DMSO in hematopoietic differentiation of hESC was investigated by using a established stepwise hematopoietic differentiation system from hESC, immunofluorescouse assay and flow cytometry. Furthermore, the window phase of DMSO action was explored by adding it to the different stage of hematopoietic differentiation. Immunofluorescence and flow cytometry analysis showed that DMSO significantly promoted the generation of CD43+ hematopoietic progenitor cells (HPC). The flow cytometry demonstrated that DMSO profoundly promoted the generation of APLNR+ lateral plate mesoderm cells and CD31+CD34+ hemogenic endothelium progenitors (HEP). The addition of DMSO in the window phase of lateral plate mesoderm cell generation could markedly improve the generation of hematopoietic progenitor cells. DMSO promotes hematopoietic differentiation of hESC through enhancing the generation of APLNR positive lateral plate mesoderm cells. The addition of DMSO in the window phase of lateral plate mesoderm cell generation can significantly improve the generation of hematopoietic progenitor cells.

SOX7 expression is critically required in FLK1-expressing cells for vasculogenesis and angiogenesis during mouse embryonic development

The transcriptional program that regulates the differentiation of endothelial precursor cells into a highly organized vascular network is still poorly understood. Here we explore the role of SOX7 during this process, performing a detailed analysis of the vascular defects resulting from either a complete deficiency in Sox7 expression or from the conditional deletion of Sox7 in FLK1-expressing cells. We analysed the consequence of Sox7 deficiency from E7.5 onward to determine from which stage of development the effect of Sox7 deficiency can be observed. We show that while Sox7 is expressed at the onset of endothelial specification from mesoderm, Sox7 deficiency does not impact the emergence of the first endothelial progenitors. However, by E8.5, clear signs of defective vascular development are already observed with the presence of highly unorganised endothelial cords rather than distinct paired dorsal aorta. By E10.5, both Sox7 complete knockout and FLK1-specific deletion of Sox7 lead to widespread vascular defects. In contrast, while SOX7 is expressed in the earliest specified blood progenitors, the VAV-specific deletion of Sox7 does not affect the hematopoietic system. Together, our data reveal the unique role of SOX7 in vasculogenesis and angiogenesis during embryonic development.

Abstract 6: A Novel in vivo Endothelial Hierarchy from Progenitor to Mature Endothelial Cells Reveals Key SoxF-Dependent Differentiation Process

Background: The formation of new blood vessels during adult life is often explained by angiogenesis. However, an alternate proposal now suggests that neo-vessels form from endothelial progenitors able to assemble all the intimal layers of vessel structures. Our aim was to define vessel-resident endothelial progenitors in vivo in a variety of tissues in physiological (aorta, lung) and pathological (wounds, tumors) situations. Methods and Results: Using common endothelial markers (CD34, CD31, VEGFR2) with flow cytometry, three sub-populations of endothelial cells could be identified among VE-Cadherin+ and CD45- cells. These were termed an endovascular progenitor (EVP) harboring CD31lo VEGFR2lo giving rise to an intermediate CD31intVEGFR2lo transit amplifying (TA) and a definitive differentiated (D) CD31hiVEGFR2hi population. Confirmation of these populations was demonstrated via lineage tracing using Cdh5cre ERt2 /Rosa-YFP reporter mice. Importantly, EVP cells arose from vascular resident beds that could not be transferred by bone marrow transplantation, marking their distinction for hematopoietic/myeloid origin. Furthermore, EVP displayed progenitor like status with a high proportion of cells in a quiescent cell cycle phase. Only EVP cells and not TA and D cells had self-renewal capacity as demonstrated by in vitro colony forming and transplant studies in vivo in Matrigel TM plugs in recipient mice. Through whole RNA sequencing we demonstrated that EVP cells highly expressed genes related to progenitor function such as Sox9 , Il33, Egfr and Pdfgrα, whereas D cells highly expressed genes related to differentiated endothelium including Ets1&2 , Gata2 , Cd31, Vwf and Notch . We also determined the Sox18 transcription factor as having a significant role in defining the endothelial hierarchy, which we validated through lineage-tracing using S ox18Cre ERt2 /Rosa-YFP mice. In the absence of functional SOX18/SOXF, EVP progenitors were still present, but TA and D populations were significantly reduced. Conclusion: In summary, we have demonstrated the existence of an entirely novel endothelial hierarchy, from EVP to TA to D. This has been demonstrated by the self-renewal, differentiation and molecular profiling of an EVP.

Cytokine-free directed differentiation of human pluripotent stem cells efficiently produces hemogenic endothelium with lymphoid potential

BackgroundThe robust generation of human hematopoietic progenitor cells from induced or embryonic pluripotent stem cells would be beneficial for multiple areas of research, including mechanistic studies of hematopoiesis, the development of cellular therapies for autoimmune diseases, induced transplant tolerance, anticancer immunotherapies, disease modeling, and drug/toxicity screening. Over the past years, significant progress has been made in identifying effective protocols for hematopoietic differentiation from pluripotent stem cells and understanding stages of mesodermal, endothelial, and hematopoietic specification. Thus, it has been shown that variations in cytokine and inhibitory molecule treatments in the first few days of hematopoietic differentiation define primitive versus definitive potential of produced hematopoietic progenitor cells. The majority of current feeder-free, defined systems for hematopoietic induction from pluripotent stem cells include prolonged incubations with various cytokines that make the differentiation process complex and time consuming. We established that the application of Wnt agonist CHIR99021 efficiently promotes differentiation of human pluripotent stem cells in the absence of any hematopoietic cytokines to the stage of hemogenic endothelium capable of definitive hematopoiesis.MethodsThe hemogenic endothelium differentiation was accomplished in an adherent, serum-free culture system by applying CHIR99021. Hemogenic endothelium progenitor cells were isolated on day 5 of differentiation and evaluated for their endothelial, myeloid, and lymphoid potential.ResultsMonolayer induction based on GSK3 inhibition, described here, yielded a large number of CD31+CD34+ hemogenic endothelium cells. When isolated and propagated in adherent conditions, these progenitors gave rise to mature endothelium. When further cocultured with OP9 mouse stromal cells, these progenitors gave rise to various cells of myeloid lineages as well as natural killer lymphoid, T-lymphoid, and B-lymphoid cells.ConclusionThe results of this study substantiate a method that significantly reduces the complexity of current protocols for hematopoietic induction, offers a defined system to study the factors that affect the early stages of hematopoiesis, and provides a new route of lymphoid and myeloid cell derivation from human pluripotent stem cells, thus enhancing their use in translational medicine.

Extracellular vesicles: emerging mediators of intercellular communication and tumor angiogenesis.

Rapidly expanding solid tumors demand large quantities of nutrients and oxygen. Growing tumors exploit pre-existing vessels and/or develop new vessels (vascularization) to obtain these nutrients. Vascularization is classified into two main processes, vasculogenesis and angiogenesis. Vasculogenesis is the formation of primitive blood vessels by endothelial progenitors, and angiogenesis encompasses the subsequent remodeling processes, including growth and migration of endothelial cells, sprouting, and stabilization of these sprouts by mural cells (1,2). Tumor cells utilize these processes to satisfy their needs and grow (tumor angiogenesis), especially under hypoxic conditions. One of the pro-angiogenic mechanisms exploited by hypoxic cancers is hypoxia-inducible transcriptional factor (HIF)-mediated signaling. Hypoxia leads to stabilization and nuclear translocation of HIF-1α, and subsequently increases transcription of pro-angiogenic genes, including vascular endothelial growth factor (VEGF) (3). Overexpression of HIF-1α has been reported in various solid tumors including brain, bladder, breast, colorectal, ovarian, pancreatic, and prostate cancers (4,5), and increased vascularity is a hallmark of poor prognosis.

The Role of Vistafin in Diabetes-Induced Impairment of Endothelial Repair System

Type 2 diabetes mellitus (T2DM) is considered a leading factor of cardiovascular (CV) events in general population. Impairment of endogenous endothelial repair system plays a pivotal role in T2DM-induced microangiopathy and macroangiopathy and, however, may contribute in CV risk and outcomes. Recent studies have shown that the nicotinamide phosphoribosyl transferase (NAMPT) currently known as visfatin has reported an integral adipocytokine with proinflammatory and immunomodulating properties. It is suggested that visfatin/NAMPT may act as multifunctional agent with undefined effect on several targets especially endothelial cells and progenitor cells depending of metabolical status of the patients. The short commentary is discussed a possible role of visfatin/NAMPT as a biomarker of nature evolution of T2DM and as a target for therapeutic strategy aimed to improve endothelial function in T2DM patients in future.

“In vitro” osteogenic and angiogenic potential evaluation of a coculture of dental pulp stem and endothelial cells grown on the BisGMA/TEGDMA Chitlac coated thermosets

Securing an adequate blood supply for survival of cell transplants is critical for a successful outcome in tissue engineering. Moreover during regeneration of weaken teeth, which is susceptible to reinfection, fracture and loss, the teeth apical canal is open and a limited blood supply is allowed. Thus the interactions between endothelial and dental pulp progenitor stem cells are important for vascularization of regenerating tissue cells. In particular, the interplay of dental pulp stem cells and endothelial cells can enhance “in vitro” osteo/odontogenic and angiogenic potential (Dissanayaka J Endod 2012, 38,454-463) and “in vivo” ensure angiogenesis and pulp regeneration (Dissanayaka Tissue Engin Part A 2015, 3-4, 550-563). Since dental pulp microenvironment supports HUVEC survival and capillary network formation in the absence of scaffolding material and external angiogenic stimulation , “in vitro” osteogenic and angiogenic potential of dental pulp stemcells cocultured with endothelial cells grown on BisGMA/TEGDMA Chitlac coated thermosets was evaluated. Results: DPSCs were grown on BisGMA/TEGDMA Chitlac coated thermosets, a composite material used in dental restoration, in the presence of two different concentrations of endothelial cells (1:1 e 1:5) for 28 days and their metabolic activity and cytotoxic response were evaluated. MTT analysis discloses that cell metabolic activity significantly increases in the presence of endothelial cells, mainly at 21 days of culture, along with cytotoxic response, while at 28 days of culture a light cytotoxic response occurs. An increasing ALP activity is evidenced in the coculture systems up to 28 days, both in the presence and in absence of Chitlac thermosets and this evidence is further supported by Alizarin red staining, which does not detect mineralization in the early stages of differentiation, but is significantly increased at 28 days of culture in both the conditions (1:1 e 1:5). Even though the positive effect on DPSC differentiation, Chitlac thermosets could induce an inflammatory response in the system and thus an ELISA IL6 assay reveals an increased inflammatory response in 1:1 coculture system after 28 days of culture, furtherly increased in 1:5 coculture system. In parallel an increased PGE2 release is evidenced in 1:1 coculture system in the presence of thermosets, reduced in 1:5 coculture system, suggesting the potential occurrence of neoangiogenesis, furtherly supported by a tubular network formation when DPSC are grown on matrigel. These results evidencing that endothelial cells enhance “in vitro” osteo/odontogenic differentiation of DPSCs and angiogenesis, and that this response is revealed in the presence of Chitlac, usually used in dental restorative practice, indicate a coculture of DPSC and endothelial cells as a promising source for regenerative endodontics.

Hemangioblast, hemogenic endothelium, and primitive versus definitive hematopoiesis.

The types of progenitors generated during the successive stages of embryonic blood development are now fairly well characterized. The terminology used to describe these waves, however,can still be confusing. What is truly primitive? What is uniquely definitive? These questions become even more challenging to answer when blood progenitors are derived invitro upon the differentiation of embryonic stem cells or induced pluripotent stem cells. Similarly, the cellular origin of these blood progenitors can be controversial. Are all blood cells, including the primitive wave, derived from hemogenic endothelium? Is the hemangioblast an invitro artifact or is this mesoderm entity also present in the developing embryo? Here, we discuss the latest findings and propose some consensus relating to these controversial issues.

Oral Mucosa Harbors a High Frequency of Endothelial Cells: A Novel Postnatal Cell Source for Angiogenic Regeneration.

Endothelial progenitor cells/endothelial cells (EPCs/ECs) have great potential to treat pathological conditions such as cardiac infarction, muscle ischemia, and bone fractures, but isolation of EPC/ECs from existing cell sources is challenging due to their low EC frequency. We have isolated endothelial progenitor (EP)-like cells from rat oral mucosa and characterized their yield, immunophenotype, growth, and in vivo angiogenic potential. The frequency of EP-like cells derived from oral mucosa is thousands of folds higher than EPCs derived from donor-match bone marrow samples. EP-like cells from oral mucosa were positive for EC markers CD31, VE-Cadherin, and VEGFR2. Oral mucosa-derived EP-like cells displayed robust uptake of acetylated low-density lipoprotein and formed stable capillary networks in Matrigel. Subcutaneously implanted oral mucosa-derived EP-like cells anastomosed with host blood vessels, implicating their ability to elicit angiogenesis. Similar to endothelial colony-forming cells, EP-like cells from oral mucosa have a significantly higher proliferative rate than human umbilical vein endothelial cells. These findings identify a putative EPC source that is easily accessible in the oral cavity, potentially from discarded tissue specimens, and yet with robust yield and potency for angiogenesis in tissue and organ regeneration.

Endothelium in the pharyngeal arches 3, 4 and 6 is derived from the second heart field

Oxygenated blood from the heart is directed into the systemic circulation through the aortic arch arteries (AAAs). The AAAs arise by remodeling of three symmetrical pairs of pharyngeal arch arteries (PAAs), which connect the heart with the paired dorsal aortae at mid-gestation. Aberrant PAA formation results in defects frequently observed in patients with lethal congenital heart disease. How the PAAs form in mammals is not understood. The work presented in this manuscript shows that the second heart field (SHF) is the major source of progenitors giving rise to the endothelium of the pharyngeal arches 3 – 6, while the endothelium in the pharyngeal arches 1 and 2 is derived from a different source. During the formation of the PAAs 3 – 6, endothelial progenitors in the SHF extend cellular processes toward the pharyngeal endoderm, migrate from the SHF and assemble into a uniform vascular plexus. This plexus then undergoes remodeling, whereby plexus endothelial cells coalesce into a large PAA in each pharyngeal arch. Taken together, our studies establish a platform for investigating cellular and molecular mechanisms regulating PAA formation and alterations that lead to disease.

Pathological Heterotopic Ossifications in Brain or Spinal Cord Injured Patients: From Ectopic Bone to Hematopoietic Stem Cell Niche Development

Heterotopic ossification (HO) is a pathology characterized by the formation of ectopic bone in soft tissues generally in muscles surrounding joints such hip, knee, elbow or shoulder. The etiology of this pathology is not known but it is well established that the risk of HO is increased after traumatic brain or spinal cord injuries and also after fractures or burns. Patients with HOs suffer from pain and the range of motion from limbs with ectopic bone is highly reduced. Currently, the only effective treatment for HOs is surgery but recurrences are frequent underpinning that a better understanding of its pathophysiology is required to cure patients.Hematopoietic marrow-like tissue is frequently observed on histological section of HO. Therefore, we investigated whether HOs from patients with traumatic brain or spinal cord injury represent an ectopic model of hematopoietic stem cell (HSC) niche as defined by the presence of HSC-supportive stromal cells.HOs were obtained from patients after surgical resection in Garches Hospital (France) with their informed consent. HOs were cut in small pieces and hematopoietic as well as endothelial cells were extracted from the HO marrow by gentle washing. Mononuclear cells were then recovered by ficoll gradient and were either directly analyzed by flow cytometry or purified by immunomagnetic selection for in vitro and in vivo experiments. Mesenchymal stromal cells (MSCs) were obtained by plastic adherence from HO explants.We first confirmed the presence of hematopoietic cells from different lineages in HO marrow such as neutrophils, monocytes/macrophages, B and T lymphocytes as evidenced by membrane expression of CD45, CD15, CD11/CD14, CD19, CD3/CD4 and CD3/CD8 antigens. HSC presence within HO marrow was further demonstrated by: (i) in vitro clonogenic assays, (ii) presence of quiescent CD45+CD34+CD38- cells with a side-population phenotype and, (iii) in vivo human hematopoietic reconstitution assays in immunodeficient NSG mice.We also show that human HO marrow contained functional MSCs and endothelial progenitors (EPs) as demonstrated by phenotypic and functional analyses. MSC expressing the classical mesenchymal markers CD73, CD90, CD105 and capable to in vitro and in vivo differentiate into osteoblasts, adipocytes and chondrocytes could be isolated from human HOs. These MSCs were also able to support in vitro long term human hematopoiesis and in vivo murine hematopoiesis when seeded on hydroxyapatite scaffolds and implanted into nude mice. Endothelial progenitors with a characteristic CD31+CD144+CD34+CD45-CD90+ phenotype were detected in the mononuclear cell fraction from HO marrows. After sorting, these cells were able to form colonies on plastic and vascular networks when cultured in a Matrigel and to express high levels of VCAM-1 and ICAM-1 adhesion molecules after TNFα stimulation, confirming their EP functional capability.In conclusion, we show for the first time that human HOs contain a marrow tissue with quiescent HSC that can proliferate and differentiate within a suitable and functional mesenchymal and endothelial microenvironment, acknowledging that HO marrows are ectopic HSC niches. We have recently demonstrated the involvement of macrophages in the development of neurogenic HOs in spinalized mice1. Taking into account the role of immune cells and the neurogenic origin of human HOs, our results bring new evidences for the role of interactions between bone and neuro-immune systems in the initiation and development of adult hematopoiesis. Mechanisms underlying these processes are currently studied in our laboratory.1 Genet F et al., J Pathol. 2015 Jun;236(2):229-40 DisclosuresNo relevant conflicts of interest to declare.

Proliferation, differentiation and migration of SCA1−/CD31− cardiac side population cells in vitro and in vivo

BackgroundSide-population (SP) cells, identified by their capacity to efflux Hoechst dye, are highly enriched for stem/progenitor cell activity. They are found in many mammalian tissues, including mouse heart. Studies suggest that cardiac SP (CSP) cells can be divided into SCA1+/CD31−, SCA1+/CD31+ and SCA1−/CD31− CSP subpopulations. SCA1+/CD31− were shown to be cardiac and endothelial stem/progenitors while SCA1+/CD31+ CSP cells are endothelial progenitors. SCA1−/CD31− CSP cells remain to be fully characterized. In this study, we characterized SCA1−/CD31− CSP cells in the adult mouse heart, and investigated their abilities to proliferate, differentiate and migrate in vitro and in vivo. Methods and resultsUsing fluorescence-activated cell sorting, reverse transcriptase/polymerase chain reaction, assays of cell proliferation, differentiation and migration, and a murine model of myocardial infarction we show that SCA1−/CD31− CSP cells are located in the heart mesenchyme and express genes characteristic of stem cells and endothelial progenitors. These cells were capable of proliferation, differentiation, migration and vascularization in vitro and in vivo. Following experimental myocardial infarction, the SCA1−/CD31− CSP cells migrated from non-infarcted areas to the infarcted region within the myocardium where they differentiated into endothelial cells forming vascular (tube-like) structures. We further demonstrated that the SDF-1α/CXCR4 pathway may play an important role in migration of these cells after myocardial infarction. ConclusionsBased on their gene expression profile, localization and ability to proliferate, differentiate, migrate and vascularize in vitro and in vivo, we conclude that SCA1−/CD31− CSP cells may serve as endothelial progenitor cells in the adult mouse heart.

Priming of endothelial colony-forming cells in a mesenchymal niche improves engraftment and vasculogenic potential by initiating mesenchymal transition orchestrated by NOTCH signaling.

The prospect of using endothelial progenitors is currently hampered by their low engraftment upon transplantation. We report that mesenchymal stem/stromal cells (MSCs), independent of source and age, improve the engraftment of endothelial colony forming cells (ECFCs). MSC coculture altered ECFC appearance to an elongated mesenchymal morphology with reduced proliferation. ECFC primed via MSC contact had reduced self-renewal potential, but improved capacity to form tube structures in vitro and engraftment in vivo Primed ECFCs displayed major differences in transcriptome compared to ECFCs never exposed to MSCs, affecting genes involved in the cell cycle, up-regulating of genes influencing mesenchymal transition, adhesion, extracellular matrix. Inhibition of NOTCH signaling, a potential upstream regulator of mesenchymal transition, in large part modulated this gene expression pattern and functionally reversed the mesenchymal morphology of ECFCs. The collective results showed that primed ECFCs survive better and undergo a mesenchymal transition that is dependent on NOTCH signaling, resulting in significantly increased vasculogenic potential.-Shafiee, A., Patel, J., Wong, H. Y., Donovan, P., Hutmacher, D. W., Fisk, N. M., Khosrotehrani, K. Priming of endothelial colony-forming cells in a mesenchymal niche improves engraftment and vasculogenic potential by initiating mesenchymal transition orchestrated by NOTCH signaling.

Abstract 244: Paclitaxel increases the assembly and function of the tumor microenvironment of metastasis in breast cancer

Abstract Chemotherapy induces influx of bone marrow-derived progenitors such as mesenchymal stem cells, endothelial progenitors and proangiogenic monocytes into the primary tumor to promote angiogenesis. Thus it is feared that chemotherapy may potentiate tumor cell invasion and metastasis. Here, we show that paclitaxel delays tumor growth in several mammary carcinoma mouse and human breast cancer models, yet it significantly increases the density of microanatomical sites called “tumor microenvironment of metastasis” (TMEM) that are responsible for tumor cell intravasation and dissemination of breast cancer. The TMEM site consists of a Mena-overexpressing cancer cell in direct contact with a Tie2hi/VEGFhi macrophage and an underlying endothelial cell. Mice treated with paclitaxel have significantly more circulating tumor cells (CTCs) and metastatic foci when compared to vehicle-treated animals indicating that the chemotherapy-induced TMEM are active in assisting tumor cell intravasation. Moreover, syngeneic transplantation of Dendra2+/PyMT tumors into FVB recipients showed significantly higher incidence of Dendra2+ cells in the lung, following paclitaxel administration. In parallel experiments, paclitaxel induced the influx of macrophages and intravasation of cancer cells as observed using intravital imaging of MMTV-PyMT-Dendra2/Cfms-CFP mice, in which blood vessels were visualized with Quantum dots. Furthermore, paclitaxel treatment in experimental mice caused a significant increase in the expression of Mena at the gene and protein levels. PCR assays for total Mena (PanMena) or specific Mena isoforms (MenaINV, Mena11a) revealed that this increase was particularly attributed to the invasive Mena isoforms [i.e. MenaINV and MenaCalc (Menacalc = PanMena - Mena11a)]. These pre-clinical data are supported by the findings from a cohort of 10 breast cancer patients who received neoadjuvant dose-dense paclitaxel followed by doxorubicin/ cyclophosphamide. Of these tumors, 7/10 patients had more than 2-fold increase in TMEM density following neoadjuvant chemotherapy regimen. Moreover, chemotherapy produced an acute increase of up to 150-fold in MenaINV expression in 3/7 and up to 5.5-fold in MenaCalc in 3/4 patients who underwent serial fine needle aspiration (FNA) biopsy before and after 1-2 doses of either neoadjuvant paclitaxel or doxorubicin-cyclophosphamide. This is provocative because an increase in either MenaCalc score or TMEM density are independently associated with increased risk of distant recurrence in breast cancer patients. In conclusion, our data indicate that paclitaxel treatment induces intravasation-mediated dissemination of breast cancer cells in rodents and in certain clinical scenarios in humans by promoting increases in MenaCalc expression and TMEM intravasation sites. Citation Format: George S. Karagiannis, Allison H. Harney, Yarong Wang, Jessica Pastoriza, Jeanine Pignatelli, Jesus Anampa, Joseph A. Sparano, Joan G. Jones, David Entenberg, John S. Condeelis, Maja H. Oktay. Paclitaxel increases the assembly and function of the tumor microenvironment of metastasis in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 244.

Abstract 1460: Bone marrow-derived CD11b+/Podoplanin+ cells are lymphatic progenitors directly responsible for breast cancer lymphatic formation

Abstract Introduction: Lymphatic metastasis, a key factor for poor outcome of breast cancer (BC), strongly depends on lymphatic vessel (LV) formation. Recent studies suggest that lymphangiogenesis is strongly promoted by bone marrow (BM)-derived CD11b+ monocytes that differentiate into Lymphatic Endothelial Cell Progenitors dubbed here M-LECP. While BC are known to recruit BM monocytes, a specific BM subset responsible for tumor lymphatic formation has not been identified. We recently discovered that podoplanin (Pdpn) is the highest upregulated lymphatic marker that signifies transdifferentiation of either human or mouse immature myeloid cells into M-LECP. We hypothesized that BM-derived M-LECP are represented by a subset expressing podoplanin in CD11b+ myeloid cells that are capable of inducing tumor lymphatics. Methods: Tumor lymphatic vessels expressing myeloid markers were detected immunohistochemically by triple-staining in clinical specimens and experimental breast tumors. Expression of Vegfr-3, Lyve-1, Sca-1, and other markers in BM-derived CD11b+/Pdpn+ and Pdpn-negative subsets was determined by FACS. Expression of lymphatic-specific markers in CD11b+/Pdpn+ and Pdpn− myeloid cells isolated from MDA-MB-231 tumors was determined by RT-qPCR. CD11b+/Pdpn+ and Pdpn-negative subsets from BM of GFP-expressing mice were adoptively transferred to mice with orthotopic breast tumors followed by quantifying mobilized GFP+/CD11b+/Pdpn+ cells and the density of tumor LV. Results: M-LECP were absent in normal human breast tissues but highly present in tumors. Analysis of clinical BC specimens showed significant correlations between tumor-mobilized M-LECP, density of LV and metastasis to regional nodes. All examined BC models exhibited very high densities (60-90%) of double-positive macrophages expressing lymphatic markers, and lymphatic vessels expressing myeloid proteins. At least a half of CD11b+ cells isolated from tumors were positive for podoplanin and nearly 90% of CD11b+/Pdpn+ subset expressed markers of progenitor cells. Only CD11b+/Pdpn+ subset (but not other myeloid cells) expressed a broad panel of proteins specific to the lymphatic endothelial lineage. Adoptive transfer of BM-derived CD11b+/Pdpn+ or Pdpn-negative fractions into tumor-bearing mice showed that only Pdpn+ BM myeloid cells integrated into preexisting LV and caused a 10-fold increase in peritumoral and intratumoral LV density. Conclusion: We show for the first time in clinical BC samples that tumor-recruited M-LECP significantly correlate with LN metastasis. We also identified a phenotypically distinct BM fraction of CD11b+/Pdpn+ cells that directly promote generation of new tumor lymphatic vessels. Our data suggest that tumors induce myeloid BM cells to undergo differentiation into M-LECP that subsequently promote cancer lymphatics thereby promoting tumor spread. Citation Format: Caitlin Griggs, Kelly Hall, Lisa Volk-Draper, Kathy Robinson, Sophia Ran. Bone marrow-derived CD11b+/Podoplanin+ cells are lymphatic progenitors directly responsible for breast cancer lymphatic formation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1460.

Cancer stem cell-still an enigma in CTCL?

Abstract Cancer stem cells have created great excitement in the research community. The concept that tumours are maintained by their own stem cells has revolutionized cancer research since the 70's. At this decade mutation in oncogenes and tumour suppressor genes were found to cause most human cancers. This led to formulating a clonal evolution concept, stating "it is proposed that most neoplasms arise from a single cell of origin, and tumour progression results from a quiet genetic viability within the original clone, allowing sequential selection of a more aggressive sub-line" [1]. This article is protected by copyright. All rights reserved.