Endothelial Cell-selective Adhesion Molecule Expression Research Articles

Endothelial Cell-Selective Adhesion Molecule Enhances Tumor Growth in a HFpEF Mouse Model

Accumulating epidemiological evidence shows that the incidence of multiple types of cancer, such as melanoma and GI cancers, is significantly higher in patients with heart failure with preserved ejection fraction (HFpEF). However, the mechanistic relationships between HFpEF and cancer development are poorly understood. Impaired angiogenesis, upregulation of endothelial adhesion molecules and chronic low-grade inflammation are common in HFpEF. Here we examined the role of endothelial cell-selective adhesion molecule (ESAM) in the progression of malignant melanoma in a HFpEF mouse model. Orthotopic melanoma xenografts (B16F10-Luc2) were used and implanted subcutaneously in mice underwent uninephrectomy and aldosterone infusion (UNX-Aldo with 1% high salt drinking water for 4 weeks) and tumor growth was monitored with bioluminescent imaging. We found that ESAM expression and serum levels were elevated in the UNX-Aldo mice, which was accompanied by enhanced melanoma growth and metastasis rate. ESAM promoted tube formation and increased phospho-Akt, phospho-PKCα and phospho-ERK1/2 in cultured endothelial cells. Flow cytometry analysis showed elevated number of tumor-associated neutrophils (CD11b+Ly6G+Ly6C-) and myeloid-derived suppressor cells (MDSCs, CD11b+F4/80-Gr-1+) in the UNX-Aldo mice. UNX-Aldo mice with genetic deletion of ESAM (ESAM KO) showed a reduced tumor growth and reduced number of tumor-associated neutrophils and MDSCs. Collectively, our results indicate that in HFpEF, a proinflammatory ESAM activation may act to promote tumor angiogenesis, neutrophil and MDSCs tumor infiltration, thereby mitigating a proper immune response and facilitate tumor growth. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Variable Expression Levels of the Hematopoietic Stem Cell Surface Antigen ESAM Depict Heterogeneity and Fluctuations in Leukemic Stem Cells

Traditionally, hematopoietic stem cells (HSCs) have been regarded as a homogeneous cell population. However, many studies have shown that populations of HSCs are essentially heterogenous. Moreover, several recent studies have proposed that HSCs exhibit fluctuating biological characteristics. Although the physiological significance of this heterogeneity and fluctuation is unclear, we presume that these qualities enable HSCs to flexibly demonstrate their self-renewal or differentiation abilities dependent on physiological demands. It is, therefore, unsurprising that such heterogeneity and fluctuation is also seen in leukemic stem cells (LSCs), and that they are involved in the development of resistance to treatment.We have previously demonstrated that the endothelial-cell selective adhesion molecule (ESAM) is an important marker of HSCs in humans and mice (Blood 2009, Exp Hematol 2013), and that the expression level of this molecule indicates the activation status of HSCs (J Immunol 2012). Furthermore, we found that a subset of leukemic cells exhibit elevated levels of ESAM expression on their surfaces (Exp Hematol 2013). Approximately two-thirds of acute myelogenous leukemia cases in humans were found to be positive for ESAM expression. Since ESAM was found to be useful in monitoring the fluctuating status of HSCs after 5-FU treatment, we posited that the marker could be used to characterize LSCs.In this study, we used ESAM expression levels as an index to analyze the heterogeneity and fluctuation in LSC populations. Flow cytometric analysis of the human leukemia cell lines KG1a and CMK revealed wide-ranging heterogeneity in ESAM expression levels, with up to a 1000-fold difference in variation, even among individual cells derived from the same clone. When these cell populations were roughly fractionated into ESAM-high or ESAM-low expression groups, the ESAM-high expression group showed a higher proliferative capacity in culture. Interestingly, however, the expression levels of ESAM were not maintained indefinitely at the same level in these groups. In four weeks, both ESAM-high and ESAM-low cell fractions developed heterogeneous ESAM expression distributions similar to that in the parent cell lines. Furthermore, when single cells were sorted and cultured, each cell showed a self-renewal ability and the heterogenous nature of ESAM expression levels was reconstructed in each population derived from these single cells. These results suggest that populations of LSCs develop heterogeneity and have a fluctuating nature.When the transcriptomes of ESAM-low and ESAM-high KG1a cells were compared using RT-PCR, the expression of the transforming growth factor beta receptor type 2 (TGFBR2) gene was found to be higher in the ESAM-low cells. Reportedly, the transforming growth factor beta (TGFβ) signaling pathway is involved in various biological processes such as cell proliferation, differentiation, and migration. In tumor cells, this pathway is involved in reducing proliferation and accelerating invasion. Based on this information, it was considered likely that the TGFβ signaling pathway is involved in the heterogeneity of ESAM expression. When TGFβ1 (5 ng/ml) was added to a culture of KG1a cells (grown in RPMI 1640 medium with 10% fetal bovine serum (FBS)), the heterogeneity of ESAM expression in the population became more pronounced, mainly due to a decrease in ESAM expression in some cells.A transcriptomic analysis of cells exhibiting decreased ESAM expression after TGFβ1 stimulation showed that these cells had a higher expression of TGFBR2 than cells not exhibiting a decrease in ESAM expression. Since the presence of 10% FBS in the culture medium provides natural TGFβ1 at a concentration of ~1 ng/ml, we conducted culture experiments without FBS. When KG1a cells were cultured in serum-free medium, the ESAM-low fraction disappeared and the heterogeneity of ESAM expression could not be replicated. However, the heterogeneity of ESAM expression in KG1a cultures could be replicated in serum-free medium by the addition of 1 ng/ml TGFβ1. These results suggest that the TGFβ signaling pathway is involved in inducing heterogeneity in ESAM expression in cell populations. The fluctuation of ESAM expression levels could be caused by differences in TGF sensitivity, which in turn, may confer certain benefits to LSCs, such as drug resistance and promoting invasiveness. DisclosuresYokota:Celgene: Research Funding; MSD K.K.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Pfizer Inc.: Research Funding; Shionogi & Co., Ltd.: Research Funding. Ezoe:Taiho Pharmaceutical Co., LTD.: Research Funding. Shibayama:Novartis Pharma K.K.: Honoraria, Research Funding; Bristol-Meyer Squibb K.K.: Honoraria, Research Funding; Ono Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Jansen Pharmaceutical K.K.: Honoraria; Fujimoto Pharmaceutical Co.: Honoraria, Research Funding; Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding. Oritani:MOCHIDA PHARMACEUTICAL: Speakers Bureau; Celgene: Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis Pharma: Consultancy, Speakers Bureau. Kanakura:Kyowa Hakko Kirin: Research Funding; Chugai Pharmaceutical: Research Funding; Pfizer: Research Funding; Bristol Myers: Research Funding; Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding; Fujimotoseiyaku: Research Funding; Eisai: Research Funding; Astellas: Research Funding; Nippon Shinyaku: Research Funding; Toyama Chemical: Research Funding; Shionogi: Research Funding.

Endothelial Cell-Selective Adhesion Molecule (ESAM) Plays Important Roles in the Adult-Type Hemoglobin Synthesis during Fetal Erythropoiesis

Endothelial cell-selective adhesion molecule (ESAM) is a lifelong marker for HSCs and is highly expressed on the first definitive HSCs. In addition, ESAM expression is functionally essential for adult HSCs because ESAM deficiency causes life-threatening myelo-suppression, especially severe anemia, after administration of 5-FU. In the last ASH annual meeting, we reported that ESAM deletion perturbed the development of definitive hematopoiesis, especially definitive erythropoiesis in the fetal liver (FL). In this study, we precisely analyzed the mechanism via which ESAM deletion affects erythroid lineage development.Although the number and the size of ESAM homozygous (homo) knockout (KO) fetuses were comparable to that of wild type (WT) or heterozygous KO littermates until embryonic day (E) 14.5, some ESAM homo KO fetuses exhibited severe anemia after E15.5 and approximately half of the ESAM homo KO fetuses died before E17.5. Quantitative real-time PCR analyses for assessing the expression of erythropoiesis-related genes in E16.5 ESAM-null FLs revealed significant reduction in mRNA levels for adult globins (Hba and Hbb-b1) and the erythroid-specific isoenzyme of 5-aminolevulinic acid synthase 2 (Alas2). In contrast, transcript levels of erythropoietin (Epo), Epo-receptor, and several transcription factors essential for erythropoiesis, such as Gata1, Klf1, and c-myb, were not reduced by ESAM deletion.Although no apparent malformation was evident in the E14.5 ESAM-null fetuses, the developmental abnormality in erythropoiesis already existed, because the proportion of enucleated erythrocytes was smaller and the number of mature Ter119+ erythroid cells was significantly decreased.Next, we examined the quality of ESAM deficient HSCs in culture and in vivo transplantation. In methylcellulose cultures, although E14.5 LSK CD48- FL cells of ESAM-null mice produced sufficient numbers of BFU-E colonies, the mRNA expression levels of adult globins and Alas2 in the BFU-E colonies produced by ESAM-null HSCs were significantly reduced. In competitive repopulation assays, 4 × 102 LSK CD48- HSCs sorted from CD45.2+ E14.5 ESAM-null or WT FLs were transplanted into lethally irradiated CD45.1+ congenic WT mice with 2 × 105 CD45.1+ bone marrow cells and analyzed after 15 weeks. Although donor chimerism and the number of CD45.2+ HSCs did not differ between the two groups, peripheral blood hemoglobin level in ESAM-null HSC-transplanted recipient mice was significantly reduced.Next, to understand the molecular mechanisms involved in the developmental failure of definitive erythropoiesis in the ESAM-null FL, we conducted RNA-sequencing (RNA-seq) and compared the gene expression patterns of WT and ESAM-null HSCs. We found that the expression of many erythropoiesis-related genes was reduced in ESAM-null HSCs. Among them, the reduction of ALAS2 expression was most remarkable. In addition, transcripts for both adult globins (Hba-a1, Hba-a2 and Hbb-b1) and embryonic globins (Hbb-y and Hbb-bh1) were dramatically decreased. These results corroborated our hypothesis that hemoglobin synthesis is impaired by ESAM deficiency, and further suggested that the cause of developmental failure in the ESAM-null fetuses can be attributed to aberrant gene expression in ESAM-null “definitive” HSCs.We exploited an antibody cross-linking method to address whether ESAM directly affected gene expression profiles in HSCs. WT E14.5 LSK CD48- cells were either untreated or incubated with a rat mAb against mouse ESAM. Then, both cells were incubated with goat anti-rat IgG Abs-conjugated microbeads for 4 h, and were subsequently applied to RNA-seq. The IPA network analysis indicated that “Hematological System Development and Function, Increased Levels of Hematocrit, Molecular Transport” network, which were associated with hemoglobin synthesis including Hbb-bh1, were the most affected. We also found up-regulation of GDF15, which promotes hemoglobin synthesis by suppressing hepcidin secretion.In conclusion, we showed that approximately half of ESAM-null fetuses died between E15.5 and E17.5, mainly due to the disturbance of adult-type hemoglobin synthesis. Although indirect mechanisms could be involved in the anemic phenotype of ESAM-null fetuses, we propose that ESAM-mediated interactions directly modulate the expression pattern of erythropoiesis-related genes in the developing HSCs. DisclosuresYokota:MSD K.K.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Pfizer Inc.: Research Funding; Shionogi & Co., Ltd.: Research Funding; Celgene: Research Funding. Ezoe:Taiho Pharmaceutical Co., LTD.: Research Funding. Shibayama:Fujimoto Pharmaceutical Co.: Honoraria, Research Funding; Jansen Pharmaceutical K.K.: Honoraria; Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Bristol-Meyer Squibb K.K.: Honoraria, Research Funding; Ono Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Novartis Pharma K.K.: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding. Oritani:Celgene: Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; MOCHIDA PHARMACEUTICAL: Speakers Bureau; Novartis Pharma: Consultancy, Speakers Bureau. Kanakura:Astellas: Research Funding; Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding; Bristol Myers: Research Funding; Nippon Shinyaku: Research Funding; Kyowa Hakko Kirin: Research Funding; Eisai: Research Funding; Chugai Pharmaceutical: Research Funding; Fujimotoseiyaku: Research Funding; Pfizer: Research Funding; Shionogi: Research Funding; Toyama Chemical: Research Funding.

Autonomous TGFβ signaling induces phenotypic variation in human acute myeloid leukemia

Heterogeneity of leukemia stem cells (LSCs) is involved in their collective chemoresistance. To eradicate LSCs, it is necessary to understand the mechanisms underlying their heterogeneity. Here, we aimed to identify signals responsible for heterogeneity and variation of LSCs in human acute myeloid leukemia (AML). Monitoring expression levels of endothelial cell‐selective adhesion molecule (ESAM), a hematopoietic stem cell‐related marker, was useful to detect the plasticity of AML cells. While healthy human hematopoietic stem/progenitor cells robustly expressed ESAM, AML cells exhibited heterogeneous ESAM expression. Interestingly, ESAM− and ESAM+ leukemia cells obtained from AML patients were mutually interconvertible in culture. KG1a and CMK, human AML clones, also represented the heterogeneity in terms of ESAM expression. Single cell culture with ESAM− or ESAM+ AML clones recapitulated the phenotypic interconversion. The phenotypic alteration was regulated at the gene expression level, and RNA sequencing revealed activation of TGFβ signaling in these cells. AML cells secreted TGFβ1, which autonomously activated TGFβ pathway and induced their phenotypic variation. Surprisingly, TGFβ signaling blockade inhibited not only the variation but also the proliferation of AML cells. Therefore, autonomous activation of TGFβ signaling underlies the LSC heterogeneity, which may be a promising therapeutic target for AML.

Sox17-mediated expression of adherent molecules is required for the maintenance of undifferentiated hematopoietic cluster formation in midgestation mouse embryos

Hematopoietic stem cell-containing intra-aortic hematopoietic cell clusters (IAHCs) emerge in the dorsal aorta of the aorta-gonad-mesonephros (AGM) region during midgestation mouse embryos. We previously showed that transduction of Sox17 in CD45lowc-Kithigh cells, which are one component of IAHCs, maintained the cluster formation and the undifferentiated state, but the mechanism of the cluster formation by Sox17 has not been clarified. By microarray gene expression analysis, we found that genes for vascular endothelial-cadherin (VE-cad) and endothelial cell-selective adhesion molecule (ESAM) were expressed at high levels in Sox17-transduced c-Kit+ cells. Here we show the functional role of these adhesion molecules in the formation of IAHCs and the maintenance of the undifferentiated state by in vitro experiments. We detected VE-cad and ESAM expression in endothelial cells of dorsal aorta and IAHCs in E10.5 embryos by whole mount immunohistochemistry. Cells with the middle expression level of VE-cad and the low expression level of ESAM had the highest colony-forming ability. Tamoxifen-dependent nuclear translocation of Sox17-ERT fusion protein induced the formation of cell clusters and the expression of Cdh5 (VE-cad) and ESAM genes. We showed the induction of the Cdh5 (VE-cad) and ESAM expression and the direct interaction of Sox17 with their promoter by luciferase assay and chromatin immunoprecipitation assay, respectively. Moreover, shRNA-mediated knockdown of either Cdh5 (VE-cad) or ESAM gene in Sox17-transduced cells decreased the multilineage-colony forming potential. These findings suggest that VE-cad and ESAM play an important role in the high hematopoietic activity of IAHCs and cluster formation.

MiR-7 Reduces Breast Cancer Stem Cell Metastasis via Inhibiting RELA to Decrease ESAM Expression

This study aimed to present evidence that miR-7 inhibited the metastasis of breast cancer stem cells (BCSCs) and elucidated the mechanisms that have remained unknown. The samples collected from miR-7 agomir-treated, BCSC-driven tumors were subjected to a protein array to analyze the protein expression profiles. A dual-luciferase reporter and chromatin immunoprecipitation-PCR were used to validate and evaluate the molecular expressions of interest in the collected breast cancer tissues and cell lines. miR-7 overexpression affecting metastasis of BCSCs was further evaluated in mice. The endothelial cell-selective adhesion molecule (ESAM) was highly expressed in breast cancer tissues and in BCSC-driven xenografts. Results of the dual-luciferase reporter and chromatin immunoprecipitation-PCR indicated that the miR-7 mimic reduced RELA expression by directly targeting the 3′ UTR of RELA to inhibit ESAM expression in MDA-MB-231 cells. Moreover, the expression levels of RELA, CD44, and ESAM were significantly decreased in lentivirus (Lenti)-miR-7-BCSC-driven xenografts compared with the control xenografts, accompanied with an increase in E-cadherin and a decrease in vimentin expression, as well as reduction in tumor growth and metastasis to lungs. Our data demonstrated that miR-7 overexpression reduced the metastasis of BCSCs via inhibiting ESAM, suggesting that ESAM could be a potential target for breast cancer therapy.

ESAM on the Cells of Endothelial Lineage Plays an Important Role in the Development of Definitive Hematopoiesis

Endothelial cell-selective adhesion molecule (ESAM) is lifelong marker of hematopoietic stem cells (HSCs) (Yokota Blood 2009) and endothelial cells (ECs) (Hirarta JBC 2001). Although we previously elucidated the functional importance of ESAM on HSCs in stress-induced hematopoiesis in adults (Sudo J Immunol 2012; PLoS One 2016), how ESAM affect hematopoietic development during fetal life remained unknown. To address this issue, we analyzed the fetuses of conventional ESAM knockout mice. Approximately half of the ESAM-null fetuses exhibited severe anemia and died after mid-gestation. RNA-sequence analyses exhibited downregulation of adult-type globin genes and Alas2, which encodes the first, rate-limiting enzyme in the heme biosynthesis pathway in the ESAM-null fetal liver (FL). Culture and transplantation experiments demonstrated that these abnormalities were attributable to the malfunctioning of ESAM-null HSCs. In addition, cross-linking ESAM with an anti-ESAM antibody influenced the transcription of hemoglobin synthesis-related genes in HSCs, indicating the significance of ESAMs on HSCs for the development of definitive hematopoiesis. However, it was unclear whether ESAM expression on only HSCs was critical or if ESAM expression on ECs was also involved. To examine this, we generated ESAM-flox mice using the innovative CLICK (CRISPR with lssDNA inducing conditional knockout alleles) method that was recently developed by our group (Miyasaka BMC Genomics 2018), and analyzed the results.We first tried to establish endothelial lineage-specific ESAM-conditional knockout (ESAM-cKO) mice by crossing ESAM-flox mice with Cre-ERT2-expressing mice under the control of the VE-cadherin gene promoter (Okabe Cell 2014). After peritoneally administering tamoxifen to pregnant females on embryonic day (E) 12.5, 14.5, or 15.5, the FLs were examined on E17.5. Analyses of these fetuses showed that, as observed in conventional ESAM-null mice, severe anemia occurred in approximately half of the Cdh5-BAC-CreERT2 ESAMflox/flox fetuses. However, we noticed that ESAM expression was almost completely deleted not only on ECs but also on HSCs of this model even after a single tamoxifen injection on E15.5.Next, we made use of another ESAM-cKO model obtained by crossing ESAM-flox mice with Cre recombinase-expressing mice under the control of the Vav gene promoter (Georgiades Genesis 2002). Although this Vav-Cre model has been widely used as a hematopoietic cell-specific gene knockout system, the EC lineage is affected by mouse-to-mouse variation (Joseph Cell Stem Cell 2013). Indeed, we observed that ESAM expression on FL ECs was suppressed with varying efficiency in each mouse, whereas ESAM expression on lineage negative Sca1+c-kitHi (LSK) CD48- cells was absent. Notably, the absolute number of LSK CD48- cells in FLs was positively correlated with the remaining levels of ESAM expression on ECs. Furthermore, the anemic phenotype was less severe and fetal mortality was less evident in Vav-Cre-induced ESAM-cKO fetuses. These results indicated that ESAM on ECs also play an important role in the normal development of definitive HSCs and erythrocytes in the FL. These findings were unexpected because we previously observed that the contribution of ESAM on non-hematopoietic cells to HSC maintenance was subtle in the adult bone marrow (BM) of transplantation-induced chimeric mice (Sudo PLoS One 2016). However, this contradiction might be explained by the difference between the FL and adult BM. Various types of cells have been reported to serve as the so-called “HSC-niche” in the adult BM, whereas most HSCs are located adjacent to sinusoidal ECs in the FL.In conclusion, our data strongly suggest that ESAM expression on ECs, as well as HSCs, plays an important role in the development of definitive hematopoiesis. ESAM is known to mediate cell-cell interaction through homophilic binding. The results showed that ESAM on ECs in the developing liver compensated for ESAM deficiency on HSCs to ameliorate the lethal anemic phenotype. This indicates that ESAM might interact with undetermined molecules on developing HSCs, and these molecules might have a functional overlap with ESAM. DisclosuresYokota:CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Pfizer Inc.: Research Funding; Bristol-Myers Squibb: Research Funding; MSD K.K.: Research Funding; Celgene: Research Funding. Ichii:Celgene K.K.: Speakers Bureau; Kowa Pharmaceutical Co.,LTD.: Speakers Bureau; Novartis Pharma K.K.: Speakers Bureau. Ezoe:TAIHO Phamaceutical Co., Ltd.: Research Funding. Shibayama:Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Fujimoto Pharmaceutical: Honoraria, Research Funding; Bristol-Meyer Squibb K.K: Honoraria, Research Funding; Ono Pharmaceutical Co.,LTD: Honoraria, Research Funding; Novartis Pharma K.K.: Honoraria, Research Funding; Jansen Pharmaceutical K.K: Honoraria; Mundipharma K.K.: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding. Oritani:Novartis Pharma: Speakers Bureau. Kanakura:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding.

Monitoring ESAM Expression Levels Reveals Autonomous Fluctuation of Leukemia Stem Cells By Autocrine/Paracrine Cytokine Signaling

Recently , we showed that hematopoietic stem cells (HSCs) fluctuate in a dynamic trajectory for self-renewal and lineage commitment, which regulates HSC heterogeneity (Cell Rep 2018). Although the significance of heterogeneity is difficult to interpret, it might enable HSCs to maintain a dynamic equilibrium of homeostatic hematopoiesis and to adapt promptly to stress-induced hematopoiesis based on physiological demands. We hypothesized that similar to HSCs, leukemia stem cells (LSCs) are heterogeneous with active fluctuation, which contributes to treatment resistance. Hence, this study aimed to show the fluctuation of LSCs and determine the underlying molecular mechanisms.We previously reported that the endothelial-cell selective adhesion molecule (ESAM) is useful as a life-long marker for HSCs in humans and mice (Blood 2009, Exp Hematol 2013). ESAM expression levels precisely indicated the activation status of HSCs (J Immunol 2012). Furthermore, approximately two-thirds of patients with acute myelogenous leukemia (AML) were positive for ESAM expression on leukemia cells in a heterogeneous manner. (Exp Hematol 2013). We exploited human AML lines as LSC-like cells and monitored expression levels of ESAM. Flow cytometry analyses revealed wide-ranging ESAM expression levels on KG1a and CMK lines. In KG1a cells, ESAM-high cells were concentrated in the CD34+CD38- fraction with high growth capability, whereas ESAM-low cells were less proliferative. Notably, when sorted into ESAM-high or ESAM-low subgroups, both groups reconstituted the parental heterogeneous distribution of ESAM levels in culture. Furthermore, single self-renewing LSC-like clones also reproduced the same heterogeneous population regardless of their original ESAM levels. These results suggested that LSC-like clones developed the heterogeneous AML cell population by fluctuating, which could be monitored with ESAM levels.The transcriptomes of ESAM-high and ESAM-low LSC-like cells in KG1a line were compared to obtain comprehensive information of key molecules involved in fluctuation and heterogeneity. We found that expression of T-cell immunoglobulin and mucin domain 3, reported as a reliable LSC marker for human AML (Cell Stem Cell 2010), was variable in parallel with ESAM. In addition, network analysis demonstrated that “genes related to hematopoietic progenitor cells” were activated whereas “genes related to blood cell maturation” were downregulated in ESAM-high cells. These results suggested that ESAM-high LSC-like cells were more identical to the conventional concept of LSCs than ESAM-low cells. However, expression levels of BAALC, SPP1, and ITGB7 were elevated in ESAM-low cells. BAALC encodes “brain and acute leukemia cytoplasmic (BAALC)” protein, which inhibits drug-induced apoptosis in KG1a cells (Hematology 2012). SPP1 encodes “secreted phosphoprotein 1 (SPP-1), also known as osteopontin, which induces chemo-resistance in AML cells (Life sci 2017). Furthermore, a recent paper reported that the active form of integrin □7 can be a therapeutic target of myeloma stem cells (Nat Med 2018). Thus, we infer that fluctuating to the ESAM-low stage may contribute to the refractory nature of LSCs in AML.We performed an up-streaming analysis on the transcriptome data to determine the signaling pathways involved in the fluctuation. The tumor necrosis factor alpha (TNFα) and the transforming growth factor beta (TGFβ) signaling pathways were found to affect ESAM expression levels. When TNFα or TGFβ1 was added to culture medium, the proliferation of KG1a cells was suppressed in a dose-dependent manner. Furthermore, addition of TNFα or TGFβ1 significantly suppressed ESAM expression levels on a subset of KG1a cells, which enhanced their heterogeneity. When cultured in serum-free cytokine-free medium, KG1a cells became uniform in terms of ESAM expression levels, with the ESAM-low population disappearing . Intriguingly, we found that KG1a cells produced TNFα and TGFβ1 autonomously, which induced the cell heterogeneity in an autocrine/paracrine manner. Surprisingly, blocking these signaling pathways by receptor inhibitors induced apoptosis in KG1a cells. These results suggest that autocrine/paracrine cytokine signaling plays important roles in promoting the variable and heterogeneous nature of LSCs, the inhibition of which might be a strategy for therapeutic intervention in refractory AML. DisclosuresYokota:Bristol-Myers Squibb: Research Funding; Pfizer Inc.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; MSD K.K.: Research Funding; Celgene: Research Funding. Ichii:Celgene K.K.: Speakers Bureau; Kowa Pharmaceutical Co.,LTD.: Speakers Bureau; Novartis Pharma K.K.: Speakers Bureau. Shibayama:Jansen Pharmaceutical K.K: Honoraria; Celgene K.K.: Honoraria, Research Funding; Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Ono Pharmaceutical Co.,LTD: Honoraria, Research Funding; Fujimoto Pharmaceutical: Honoraria, Research Funding; Bristol-Meyer Squibb K.K: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; Novartis Pharma K.K.: Honoraria, Research Funding. Kanakura:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding.

A Comparison of Endothelial Cell-Selective Adhesion Molecule and von Willebrand Factor Expression in Breast Cancer Growth and Metastasis

Background: Angiogenesis is the process of new blood vessels formation that contribute to tumor growth and metastasis. Endothelial cell-selective adhesion molecule is one of the proteins that expresses in vascular endothelial cells. In vitro and animal studies have shown involvement of this protein in physiological and pathological angiogenesis. von Willebrand factor is a protein expressed by endothelial cells and megakaryocytes that has a role in blood clotting processes. In the current study, we investigate the expression of endothelial cell-selective adhesion molecule and von Willebrand factor in carcinoma mammae specimens and explore their correlation with tumor growth and metastasis. Methods: We obtained 79 specimens from paraffin blocks of patients diagnosed with invasive breast carcinoma of no special type. The slides from these specimens were then stained with endothelial cell-selective adhesion molecule, von Willebrand factor, and Ki-67 antibodies to assess vascular numbers and cell proliferation. Results: We found a total of 31 (39%) low vascularity and 48 (61%) high vascularity samples from endothelial cell-selective adhesion molecule staining. There were 34 (43%) low vascularity and 45 (54%) high vascularity samples by von Willebrand factor staining. There was a significant correlation of blood vessel numbers in the endothelial cell-selective adhesion molecule-stained samples with tumor volume, metastasis to lymph nodes, and proliferation cells. The von Willebrand factor-expressed samples only had a significant correlation of vascular number with tumor volume. Conclusion: Endothelial cell-selective adhesion molecule and von Willebrand factor as the endothelial cell expressed proteins play a role in the angiogenesis process of breast cancer. However, endothelial cell-selective adhesion molecule expression is more consistent than von Willebrand factor in predicting the presence or absence of metastatic breast cancer.

Endothelial Cell-Selective Adhesion Molecule (ESAM) Is Required for the Ontogeny of Definitive Hematopoietic System in Mice

We previously reported that endothelial cell-selective adhesion molecule (ESAM), which was initially identified as an endothelial cell-specific antigen, is an effective lifelong hematopoietic stem cell (HSC) marker in mice and humans (Yokota Blood 2009; Ishibashi Exp Hematol 2016). Prior to the advent of the first definitive HSC, ESAM was already expressed on hemogenic endotherium in the developing aorta of murine embryos. We also reported that ESAM expression on HSCs is functionally important for adult hematopoiesis because ESAM deficiency causes life-threatening myelo-suppression, especially severe anemia, after administration of 5-fluorouracil (5-FU) (Sudo J Immunol 2012, PLoS One 2016). Collective data obtained from the genotyping of newborn ESAM knockout (KO) mice suggested that the number of homozygous (homo) ESAM KO mice was about half of that expected as per the Mendelian ratio. The functional significance of ESAM in the development of hematopoiesis, however, has yet to be determined. Thus, in the present study we have analyzed how ESAM deletion affects hematopoietic development in fetuses of ESAM KO mice.Unexpectedly, the frequency and the size of ESAM homo KO fetuses were comparable to those of wildtype (WT) or heterozyqous KO littermates at embryonic day (E) 14.5. However, we found that the liver of ESAM homo KO fetuses contained significantly fewer mononuclear cells. FACS analyses revealed that all the tested hematopoietic cell populations, including lineage- Sca1+cKitHigh (LSK) and LSK CD150+ CD48- HSCs, B220+ B cells, Gr1+ myeloid cells, and Ter119+ erythroid cells, were significantly decreased in the ESAM homo KO fetal liver. Erythroid differentiation was thought to be delayed in ESAM homo KO fetuses because Ter119+ mature erythroid cells significantly decreased whereas CD71+ Ter119- immature cells significantly increased. HSC-enriched LSK cells from E14.5 ESAM homo KO mice produced fewer numbers of blood cells in MS5 co-culture than those from the others, particularly B-lineage cells, suggesting that the growth and differentiation potential of HSCs is impaired in the absence of ESAM.Although ESAM-deficient fetuses grew without an apparent malfunction in developing organs until E14.5, we found that life-threatening events occurred in 3 days following E14.5. Approximately half of homo KO fetuses exhibited severe anemia at E15.5 and died before E17.5. Quantitative real-time PCR analyses from E16.5 ESAM KO homo fetal livers revealed a significant reduction in messenger RNA (mRNA) levels for adult globins (α and β major). In addition, the mRNA level for an erythroid-specific isoenzyme of 5-aminolevulinic acid synthase 2 (ALAS2), the first and rate-limiting enzyme in the heme biosynthesis pathway, was also found to be reduced in the liver of E16.5 ESAM KO homo fetuses.To learn more about molecular mechanisms involved in the developmental failure of hematopoiesis in ESAM KO fetuses, we performed RNA sequencing (RNA-seq) of LSK cells sorted from E14.5 WT and ESAM KO homo mice. We found that, while transcripts for embryonic globins (ζ and Ey) remained substantially, those for adult globins (α, β major, and β minor) were markedly down-regulated in ESAM-KO HSCs. The results suggested that ESAM deficiency disturbs the globin switch from embryonic to adult type. ALAS2 was insufficiently induced in LSK cells of ESAM KO fetal livers, which presumably results in defects in heme biosynthesis. During the embryonic development, rapid and explosive production of erythroid cells is imperative to support the growth and survival of fetuses. To meet the physiological requirement, definitive erythropoiesis occurs in the developing liver and replaces primitive erythropoiesis. Our data suggest that ESAM expression is indispensable for the development of definitive erythropoiesis.In conclusion, we have revealed that ESAM plays a critical role in the development of definitive hematopoiesis. Approximately half of ESAM KO homo fetuses died between E15.5 and E17.5, at least partly due to the delay of adult hemoglobin synthesis in the absence of ESAM. DisclosuresYokota:SHIONOGI & CO., LTD.: Research Funding. Doi:Yakult Honsha Co.,Ltd.: Speakers Bureau. Shibayama:Novartis Pharma: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding, Speakers Bureau; Takeda: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau; Ono Pharmaceutical: Speakers Bureau. Kanakura:Chugai Pharmaceutical: Research Funding; Pfizer: Research Funding; Shionogi: Research Funding; Kyowa Hakko Kirin: Research Funding; Fujimotoseiyaku: Research Funding; Toyama Chemical: Research Funding; Bristol Myers: Research Funding; Alexionpharma: Research Funding; Nippon Shinyaku: Research Funding; Astellas: Research Funding; Eisai: Research Funding.

Endothelial Cell-Selective Adhesion Molecule Expression in Hematopoietic Stem/Progenitor Cells Is Essential for Erythropoiesis Recovery after Bone Marrow Injury.

Numerous red blood cells are generated every second from proliferative progenitor cells under a homeostatic state. Increased erythropoietic activity is required after myelo-suppression as a result of chemo-radio therapies. Our previous study revealed that the endothelial cell-selective adhesion molecule (ESAM), an authentic hematopoietic stem cell marker, plays essential roles in stress-induced hematopoiesis. To determine the physiological importance of ESAM in erythroid recovery, ESAM-knockout (KO) mice were treated with the anti-cancer drug, 5-fluorouracil (5-FU). ESAM-KO mice experienced severe and prolonged anemia after 5-FU treatment compared to wild-type (WT) mice. Eight days after the 5-FU injection, compared to WT mice, ESAM-KO mice showed reduced numbers of erythroid progenitors in bone marrow (BM) and spleen, and reticulocytes in peripheral blood. Megakaryocyte-erythrocyte progenitors (MEPs) from the BM of 5-FU-treated ESAM-KO mice showed reduced burst forming unit-erythrocyte (BFU-E) capacities than those from WT mice. BM transplantation revealed that hematopoietic stem/progenitor cells from ESAM-KO donors were more sensitive to 5-FU treatment than that from WT donors in the WT host mice. However, hematopoietic cells from WT donors transplanted into ESAM-KO host mice could normally reconstitute the erythroid lineage after a BM injury. These results suggested that ESAM expression in hematopoietic cells, but not environmental cells, is critical for hematopoietic recovery. We also found that 5-FU treatment induces the up-regulation of ESAM in primitive erythroid progenitors and macrophages that do not express ESAM under homeostatic conditions. The phenotypic change seen in macrophages might be functionally involved in the interaction between erythroid progenitors and their niche components during stress-induced acute erythropoiesis. Microarray analyses of primitive erythroid progenitors from 5-FU-treated WT and ESAM-KO mice revealed that various signaling pathways, including the GATA1 system, were impaired in ESAM-KO mice. Thus, our data demonstrate that ESAM expression in hematopoietic progenitors is essential for erythroid recovery after a BM injury.

ESAM is a novel human hematopoietic stem cell marker associated with a subset of human leukemias

Reliable markers are essential to increase our understanding of the biological features of human hematopoietic stem cells and to facilitate the application of hematopoietic stem cells in the field of transplantation and regenerative medicine. We previously identified endothelial cell-selective adhesion molecule (ESAM) as a novel functional marker of hematopoietic stem cells in mice. Here, we found that ESAM can also be used to purify human hematopoietic stem cells from all the currently available sources (adult bone marrow, mobilized peripheral blood, and cord blood). Multipotent colony-forming units and long-term hematopoietic-reconstituting cells in immunodeficient mice were found exclusively in the ESAM(High) fraction of CD34(+)CD38(-) cells. The CD34(+)CD38(-) fraction of cord blood and collagenase-treated bone marrow contained cells exhibiting extremely high expression of ESAM; these cells are likely to be related to the endothelial lineage. Leukemia cell lines of erythroid and megakaryocyte origin, but not those of myeloid or lymphoid descent, were ESAM positive. However, high ESAM expression was observed in some primary acute myeloid leukemia cells. Furthermore, KG-1a myeloid leukemia cells switched from ESAM negative to ESAM positive with repeated leukemia reconstitution in vivo. Thus, ESAM is a useful marker for studying both human hematopoietic stem cells and leukemia cells.

ESAM Is a Novel Human Hematopoietic Stem Cell Marker Associated with a Subset of Human Leukemias

Murine hematopoietic stem cells (HSCs) can be isolated with high efficiency as Lineage- Sca-1+ c-kitHigh (LSK) CD34-/Low CD150+ CD48- cells. In humans, however, the same method is not useful because of critical differences between murine and human HSC phenotypes. Such discrepancy has hampered the translation of findings in mice into a human preclinical or clinical context. Therefore, the identification of common HSC antigens between the two species would be a significant advance with respect to translational studies of HSC biology. We previously identified endothelial cell-selective adhesion molecule (ESAM) as a novel maker for HSCs in mice (Blood, 2009). We also found that ESAM is functionally important for murine HSCs to reconstitute hematopoiesis after 5-FU treatment (J Immunol, 2012). In the present study, we have extended our research of ESAM to human HSCs and leukemia.We first examined whether ESAM expression showed potential as a marker of human HSCs. In addition to adult BM, the majority of CD34+ CD38- cells in cord blood (CB) and G-CSF mobilized peripheral blood expressed ESAM. The addition of anti-CD90 and CD45RA antibodies divides the adult BM CD34+ CD38- fraction into three subpopulations, namely HSCs, multipotent progenitors (MPPs), and multi-lymphoid progenitors (MLPs). We found that HSCs expressed high levels of ESAM whereas MPPs expressed lower levels and many MLPs lost ESAM expression. Functional assessment for ESAM-/Low and ESAMHigh cells in the CD34+ CD38- fractionconfirmed that high ESAM expression distinguishes progenitors that are more primitive and multipotent.We also identified a subset of CD34+ CD38- cells in adult BM and CB that expressed extremely high levels of ESAM, namely ESAMBright cells. Gene expression profiles of the CD34+ CD38- ESAMHigh and CD34+ CD38- ESAMBright populations showed that the former cells expressed HSC-related genes whereas the latter showed more endothelial-related profiles. Indeed, the CD34+ CD38- ESAMBright cells produced CD31+ endothelial cells, but not CD45+ hematopoietic cells, in co-culture with MS5 stromal cells. These results suggest that the CD34+ CD38- fraction, which is conventionally considered the human HSC fraction, also contains a substantial number of non-hematopoietic progenitors. Thus, the inclusion of ESAM provides a more accurate estimation of HSC numbers.Since some of HSC-related antigens are useful for determining leukemia lineage and have utility as prognostic indicators, we determined whether ESAM might also be a valuable addition to this antigen panel. First, we examined human leukemia cell lines. Tested myeloid leukemia lines including KG-1a, HL60, THP1, U937 and Kasumi were uniformly negative for ESAM expression. Jurkat and MOLT4, lymphoid lineage lines were also negative. On the other hand, HEL, an erythroid leukemia cell line, and CMK, a megakaryocytic leukemia cell line, exhibited high expression of ESAM. Additionally, K562 cells, which originated from CML that subsequently transformed into acute erythro-leukemia, also express ESAM. We then evaluated ESAM expression on primary acute leukemia cells, which were isolated from patients upon diagnosis. Interestingly, while all of ALL cases were virtually negative for ESAM, more than half of AML cases were ESAM-positive. Notably, the ESAM expression pattern on AML cases substantially differs even in the same FAB classification.We inferred that AML cells might change their ESAM expression levels according to cell intrinsic features and/or the surrounding environment in vivo. Therefore, we inoculated ESAM- KG-1a cells into NOD/SCID mice and harvested reconstituted KG-1a (rKG-1a) cells after the inoculation. They were then cultured in vitro and inoculated again into NOD/SCID mice. FACS analyses revealed that, although parental KG-1a cells were ESAM-negative, rKG-1a cells expressed a substantial amount of ESAM. Notably, rKG-1a cells were more aggressive and killed the recipient mice in a shorter period. This observation indicates that leukemia cells change their surface phenotype according to the environment, and that ESAM expression may be related to the acquisition of a more aggressive phenotype.In conclusion, we demonstrate that ESAM is a reliable marker of HSCs in humans as well as in mice. Additionally, ESAM is expressed on some of human acute leukemia cells and might be useful for lineage determination and as prognostic indicator. DisclosuresYokota:SHIONOGI & CO., LTD.: Research Funding. Kanakura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

O1-11-6 - Expression of Esam and Vwf as Angiogenesis Marker in Tumor Growth and Metastasis of Carcinoma

Background: The formation of new vascular (angiogenesis) is one of important step in tumor growth and have correlation with the capability of tumor cell to mestastase in distant place. Endothelial cell-Selective Adhesion Molecule (ESAM) is protein which expressed in endothelial cell of blood vessel. Von Willebrand Factor (vWF) is glycoprotein which is found in blood plasma and were produced by both endothelial cell and megakaryocytes. Meanwhile, Ki-67 is one of cell protein which is coded by MKi-67 gene in human cells and have relations with cell proliferation.Methods: A series of 79 tissue samples from carcinoma mammae were stained immunohistochemically with ESAM, vWF and Ki-67 antibodies to evaluate its vascularity and cell proliferation.Results: From these samples, it was found that 31 (39%) samples have low vascularity and 48 (61%) samples have high vascularity according to ESAM expression. Meanwhile, in correlated with vWF expression, there were 34 (43%) samples with low vascularity and 45 (54%) samples with high vascularity. There was a significant correlation of totally number of vascular in tumor samples which have stained with ESAM and vWF with totally number of cells which expressed Ki-67 and tumor size/volume. There was only a significant correlation in between totally number of vascularity which expressed ESAM and the presence of lymphe nodes metastasis. There was also significant correlation between increase of vascularity number and number of cells with Ki-67 expression.Conclusion: It was concluded that angiogenesis was highly required in tumor growth and metastases and both of ESAM and vWF have involved in angiogenesis process of breast cancer. However, ESAM looks more sensitive than vWF to predict the presence of metastases in breast cancer.

Endothelial Cell-Selective Adhesion Molecule Marks Human Hematopoietic Stem Cells Regardless Of The HSC Sources

Identification of novel markers associated with hematopoietic stem cells (HSCs) is important to progress basic and clinical research regarding the HSC biology. We previously reported that endothelial cell-selective adhesion molecule (ESAM) marks HSCs throughout life in mice (Yokota et al. Blood, 2009). We also demonstrated that ESAM can be a useful indicator of activated HSCs after bone marrow (BM) injury and that ESAM is functionally important for recovering hematopoiesis by using ESAM knockout mice (Sudo et al. J Immunol, 2012). However, the discrepancy between species has been a long-standing obstacle to apply findings in mice to human. For example, established murine HSC markers such as Sca-1 or CD150 are not expressed on human HSCs. Thus, it is important to know if ESAM marks HSCs beyond species and serves as a functional molecule for the HSC property, but information regarding ESAM expression in human HSCs has been quite limited. In this study, we have examined the ESAM expression pattern on human HSCs derived from diverse sources. In addition, we have performed functional assessment of the ESAM-expressing cells.Cord blood (CB), aspirated BM, and granulocyte-colony stimulating factor-mobilized peripheral blood (GMPB) were obtained from healthy donors. BM was also obtained from head of femora of patients who received the hip replacement surgery. All of the protocols were approved by the Institutional Review Board of Osaka University School of Medicine, and we obtained the written agreement form with informed consent from all participants. Mononuclear cells were separated using Ficoll centrifugation from CB, aspirated BM and GMPB. For preparation of BM cells adjacent to bone tissues, trabecular tissues of femora were treated with 2 mg/ml collagenase IV and DNase and gently agitated for 1 hour at 37 °C. Collected cells were analyzed using flow cytometry for cell surface expression of ESAM and other markers. Further, the CD34+ CD38−cells were fractionated according to the intensity of ESAM expression and evaluated in vivo and in vitro functional assays.Flow cytometry analyses revealed that the majority of CB CD34+ CD38− cells expressed ESAM. According to the expression level, CB CD34+ CD38− cells could be subdivided into three populations, namely ESAM−/Low, ESAMHigh, and ESAMBright. While all CB contained a robust ESAMHigh population in CD34+ CD38− cells, the percentage of ESAMBright cells varied widely among CB samples. The ESAMHigh CD34+ CD38− cells also expressed CD90 and CD133, which are known as HSC markers. Methylcellulose colony-forming assays and limiting dilution assays revealed that ESAMHigh fraction enriches primitive hematopoietic progenitors. Further, ESAMHigh cells also reconstituted the long-term human hematopoiesis in NOD/Shi-scid, IL-2Rγnull (NOG) mice. Therefore, as in mice, ESAMHighmarks authentic HSCs in human.On the other hand, ESAMBright CD34+ CD38− cells showed low colony-forming activities and no reconstitution of human hematopoiesis in NOG mice. These ESAMBright CD34+ CD38− cells expressed CD118/leukemia inhibitor factor receptor and endothelial markers such as VE-Cadherin, Flk-1, and CD146, but not CD45. These results suggested that ESAMBright cells in the CB CD34+ CD38− fraction are non-hematopoietic cells. With respect to the other HSC sources such as aspirated BM and GMPB, almost all CD34+ CD38− cells were ESAMHigh and ESAMBright cells were not found in this fraction. Interestingly, however, ESAMBright cells were found in the CD34+ CD38− fraction isolated from collagenase-treated femora. These BM-derived ESAMBright CD34+ CD38− cells expressed endothelial markers as did the CB-derived cells. They could generate CD31+endothelial cells, but not hematopoietic cells in coculture with MS5 stromal cells with vascular endothelial growth factor, stromal-cell-derived factor, and interleukin 16.In conclusion, ESAM expression serves as a marker to enrich HSCs in human regardless of the HSC sources. In addition, the very high intensity of this marker might be useful to isolate non-hematopoietic progenitors from CD34+ CD38− cells, which has been conventionally used as human HSCs. The common feature of ESAM expression of murine and human HSCs suggests a possibility that functional significance of ESAM expression obtained from mouse studies could be applicable to human. Disclosures:No relevant conflicts of interest to declare.

Low serum endothelial cell-selective adhesion molecule predicts increase in albuminuria in type 2 diabetes patients

The newly discovered endothelial cell-selective adhesion molecule (ESAM) stabilizes the interendothelial tight junction; it circulates in serum as a soluble fraction. In experimental diabetes, reduced ESAM expression in the kidney is associated with albuminuria. We investigated, for the first time, serum ESAM as a predictor of progression of kidney disease in type 2 diabetes (T2D). T2D non-nephrotic patients with glomerular filtration rate (GFR) > 30 ml/min were included. History, medication and laboratory evaluation were assessed at inclusion and the end of study; ESAM was determined at baseline. Eighty-eight patients--mean age 63 ± 10.84 years, 49 (55.68 %) males--were prospectively followed up for 20 months. Baseline GFR was 76.37 ± 29.56 ml/min, and urinary albumin/creatinine ratio (UACR) 21.63(7.08-94.52) mg/g; ESAM was 12.85(6.13-19.83) ng/ml. Difference (Δ) in UACR between end of study and baseline was inversely related to serum albumin (r = -0.27, p = 0.017) and ESAM (r = -0.21, p = 0.047); ΔGFR correlated to glycated hemoglobin (r = 0.22, p = 0.05). In multiple regression, introducing variables susceptible to influence progression of kidney disease, ΔUACR was significantly related to log ESAM (p = 0.005) and ΔGFR to glycated hemoglobin (p = 0.016). Serum ESAM is a predictor of worsening of albuminuria in T2D patients without advanced kidney disease.

The Endothelial Antigen ESAM Monitors Hematopoietic Stem Cell Status between Quiescence and Self-Renewal

Whereas most hematopoietic stem cells (HSC) are quiescent in homeostasis, they actively proliferate in response to bone marrow (BM) injury. Signals from the BM microenvironment are thought to promote entry of HSC into the cell cycle. However, it has been cumbersome to assess cycle status of viable HSC and thus explore unique features associated with division. In this study, we show that expression of endothelial cell-selective adhesion molecule (ESAM) can be a powerful indicator of HSC activation. ESAM levels clearly mirrored the shift of HSC between quiescence and activation, and it was prominent in comparison with other HSC-related Ags. ESAM(hi) HSC were actively dividing, but had surprisingly high long-term reconstituting capacity. Immunohistochemical analyses showed that most ESAM(hi) HSC were located near vascular endothelium in the BM after 5-fluorouracil treatment. To determine the importance of ESAM in the process of BM recovery, ESAM knockout mice were treated with 5-fluorouracil and their hematopoietic reconstruction was examined. The ESAM deficiency caused severe and prolonged BM suppression, suggesting that ESAM is functionally indispensable for HSC to re-establish homeostatic hematopoiesis. With respect to intracellular regulators, NF-κB and topoisomerase II levels correlated with the ESAM upregulation. Thus, our data demonstrate that the intensity of ESAM expression is useful to trace activated HSC and to understand molecular events involved in stem cell states.

The Endothelial Antigen ESAM Monitors Reversible Conversion of Hematopoietic Stem Cells Between Dormancy and Self-Renewal

Abstract 2337Although hematopoietic stem cells (HSC) are characterized with self-renewal and pluri-potential, their cell-cycle status and differentiating behavior do fluctuate according to the physiological requirement. In the homeostatic state of adult bone marrow (BM), HSC are likely to be quiescent so that they can evade exhaustion or mutation. However, when BM is injured by irradiation and/or anti-cancer drugs, HSC need to proliferate to restore normal hematopoiesis. Then, after re-establishment of homeostasis, activated HSC return to be quiescent. Molecular crosstalk between HSC and BM microenvironment is thought to elaborately control the status of HSC, but precise mechanisms remain unknown. If the conversion of HSC between dormancy and self-renewal could be accurately monitored, the method should be useful to understand how the HSC status is regulated.Our previous study demonstrated that endothelial cell-selective adhesion molecule (ESAM) is a useful marker for murine HSC throughout life. In the present study, we examined if the ESAM level reflects the HSC status between dormancy and activation. Firstly we monitored ESAM levels of the Lin− Sca1+ c-kit+ (LSK) HSC-enriched fraction in BM after a single 5-FU injection (150 mg/kg) by flow cytometry. From 2 to 9 days after the 5-FU injection, ESAM levels on the LSK fraction remarkably increased. Indeed, the mean fluorescence intensity of ESAM expression on HSC increased by 9.6-fold in 5 days after 5-FU injection. The increase of ESAM expression was more drastic than that of other endothelial-related markers such as CD34 (1.6-fold). After reaching to the maximum peak around day 5–6, ESAM level gradually decreased and returned to the homeostatic level by 12 days after 5-FU. Interestingly, the ESAM up-regulation on HSC was abrogated when inhibitory drugs for NF-kB and topoisomerase-II were given after 5-FU injection. Furthermore, short-term BrdU exposure proved that the ESAMhi cells after 5-FU treatment are actually active in the cell cycle status. Then, the immuno-histochemical analyses were performed to locate the activated HSC in 5-FU treated BM. Since more than 80% of the Lin− ESAMhi Sca1+ cells were found within 20 μm from vascular endothelium, the activated HSC seemed to be intimate with endothelial cells and/or vascular-related cells.Next, we performed functional assessments of the ESAMlow LSK and ESAMhi LSK fractions sorted from 5-FU-treated BM. In methylcellulose cultures, while both fractions contained a number of hematopoietic progenitors, CFU-Mix, primitive multipotent progenitors, were significantly enriched in the ESAMhi fraction (10±0 vs. 48.5±2.1 per 200 ESAMlow or ESAMhi LSK cells, respectively). In the in vivo long-term reconstitution assays, we transplanted 2,000 CD45.1+ ESAMlow or ESAMhi LSK cells with 2 × 105 CD45.2+ competitor BM cells into lethally irradiated CD45.2+ mice. Sixteen weeks after transplantation, the mice transplanted with ESAMhi LSK cells showed significantly higher chimerisms of CD45.1+ cells than those transplanted with ESAMlow LSK, suggesting that long-term HSC are enriched in the ESAMhi fraction. It is noteworthy that the ESAMhi CD45.1+ LSK fraction re-constituted a CD45.1+ LSK population in the CD45.2+ recipient BM, whose ESAM expression levels lowered to the homeostatic level.The results above suggested that ESAM expression level mirrors the activation status of HSC after BM injury. However, it remains unclear if ESAM plays an important role in the hematopoietic recovery. Although we did not observe significant phenotypes except slight anemia in homeostatic ESAM KO mice, we presumed that substantial BM stress might reveal physiological importance of the ESAM expression. At day 5 after injecting 200mg/kg 5-FU, we found that leukocytes and platelet were remarkably decreased in KO mice. Furthermore, the KO mice showed severe anemia (Hb; WT 10.4±1.1 g/dl vs. KO 6.0±1.7 g/dl at day 10), and two of five mice died at day 12. In addition, we observed LSK Flt3− HSC as well as total mononuclear cells more significantly decreased in the KO mice.In summary, our data have shown that ESAM serves as a strong tool to monitor the conversion between dormancy and proliferation of adult BM HSC. In addition, the data from ESAM KO mice have suggested that ESAM is indispensable for normal hematopoietic recovery after BM injury. Further studies should address physiological meanings of the high ESAM level on active HSC. Disclosures:No relevant conflicts of interest to declare.

Role of endothelial cell-selective adhesion molecule in hematogeneous metastasis

The spread of malignant cells from a localized tumor is thought to be directly related to the number of microvessels in the tumor. The endothelial cell-selective adhesion molecule (ESAM) is a member of the immunoglobulin superfamily that mediates homophilic interactions between endothelial cells. Previous studies have indicated that ESAM regulates angiogenesis in the primary tumor growth and endothelial permeability. In this study, we aimed to further elucidate the role of ESAM in tumor metastasis through angiogenic processes. ESAM expression was higher in hypervascular metastatic tumor tissues than in normal tissues in human lungs. Cell culture studies found that conditioned medium from B16F10 melanoma cells increased ESAM expression in endothelial cells and promoted endothelial migration and tube formation. The B16F10 medium-induced endothelial migration and tube formation were significantly attenuated when ESAM was downregulated by siRNA transfection. Intravenous injection of B16F10 cells into ESAM+/+ and ESAM−/− mice for comparison of metastatic potential resulted in the number of metastatic lung nodules in ESAM−/− mice being 83% lower than of those in ESAM+/+ mice. The microvascular density in the tumor was also lower in ESAM−/− than in ESAM+/+ mice. These findings indicate that ESAM regulates tumor metastasis through endothelial cell migration and tube formation in metastatic nodules. Inhibition of ESAM may therefore inhibit tumor metastasis by inhibiting the angiogenic processes.

The endothelial antigen ESAM marks primitive hematopoietic progenitors throughout life in mice

AbstractAlthough recent advances have enabled hematopoietic stem cells (HSCs) to be enriched to near purity, more information about their characteristics will improve our understanding of their development and stage-related functions. Here, using microarray technology, we identified endothelial cell-selective adhesion molecule (ESAM) as a novel marker for murine HSCs in fetal liver. Esam was expressed at high levels within a Rag1− c-kitHi Sca1+ HSC-enriched fraction, but sharply down-regulated with activation of the Rag1 locus, a valid marker for the most primitive lymphoid progenitors in E14.5 liver. The HSC-enriched fraction could be subdivided into 2 on the basis of ESAM levels. Among endothelial antigens on hematopoietic progenitors, ESAM expression showed intimate correlation with HSC activity. The ESAMHi population was highly enriched for multipotent myeloid-erythroid progenitors and primitive progenitors with lymphopoietic activity, and exclusively reconstituted long-term lymphohematopoiesis in lethally irradiated recipients. Tie2+ c-kit+ lymphohematopoietic cells in the E9.5–10.5 aorta-gonad-mesonephros region also expressed high levels of ESAM. Furthermore, ESAM was detected on primitive hematopoietic progenitors in adult bone marrow. Interestingly, ESAM expression in the HSC-enriched fraction was up-regulated in aged mice. We conclude that ESAM marks HSC in murine fetal liver and will facilitate studies of hematopoiesis throughout life.