CRLF3 plays a key role in the final stage of platelet genesis and is a potential therapeutic target for thrombocythemia.

The process of platelet production has so far been understood to be a 2-stage process: megakaryocyte maturation from hematopoietic stem cells followed by proplatelet formation, with each phase regulating the peripheral blood platelet count. Proplatelet formation releases into the bloodstream beads-on-a-string preplatelets, which undergo fission into mature platelets. For the first time, we show that preplatelet maturation is a third, tightly regulated, critical process akin to cytokinesis that regulates platelet count. We show that deficiency in cytokine receptor-like factor 3 (CRLF3) in mice leads to an isolated and sustained 25% to 48% reduction in the platelet count without any effect on other blood cell lineages. We show that Crlf3-/- preplatelets have increased microtubule stability, possibly because of increased microtubule glutamylation via the interaction of CRLF3 with key members of the Hippo pathway. Using a mouse model of JAK2 V617F essential thrombocythemia, we show that a lack of CRLF3 leads to long-term lineage-specific normalization of the platelet count. We thereby postulate that targeting CRLF3 has therapeutic potential for treatment of thrombocythemia.

Myeloid cell subpopulations compensate each other for Ccr2-deficiency in glioblastoma.

AIMS: Glioblastomas are high-grade brain tumours that are characterised by the accumulation of brain-resident microglia and peripheral macrophages. Recruitment of these myeloid cells can be facilitated by CCR2/CCL2 signalling. Besides the well-known CCR2+ macrophages, we have identified microglia expressing CCR2 in glioma tissues. Thus, we investigated how Ccr2-deficiency of one of the myeloid cell populations affects the other population and tumour biology. METHODS: We generated four chimeric groups to analyse single and combined Ccr2-deficiency of microglia and macrophages. On day 21 after tumour cell implantation (GL261), we conducted flow cytometry, immunofluorescence and real-time polymerase chain reaction analyses. Tumour volume and metabolism were determined by magnetic resonance imaging and magnetic resonance spectroscopy. Moreover, in vitro studies were performed with primary microglia and bone marrow-derived macrophages. RESULTS: We demonstrated reduced infiltration of macrophages and microglia depending on the lack of Ccr2. However, the total number of myeloid cells remained constant except for the animals with dual Ccr2-knockout. Both microglia and macrophages with Ccr2-deficiency showed impaired expression of proinflammatory molecules and altered phagocytic activity. Despite the altered immunologic phenotype caused by Ccr2-deficiency, glioma progression and metabolism were hardly affected. Alterations were detected solely in apoptosis and proliferation of tumours from animals with specific Ccr2-deficient microglia, whereas vessel stability was increased in mice with Ccr2-knockout in both cell populations. CONCLUSION: These results indicate that microglia and macrophages provide a homoeostatic balance within glioma tissue and compensate for the lack of the corresponding counterpart. Moreover, we identified that the CCR2/CCL2 axis is involved in the immunologic function of microglia and macrophages beyond its relevance for migration.

VEGF as a modulator of the innate immune response in glioblastoma.

VEGF is an important factor in tumor vascularization and used as target for anti-angiogenic treatment strategies in glioma. In this study, we demonstrate for the first time that VEGF is a modulator of the innate immune response with suppressive effects on the immunologic and pro-angiogenic function of microglia/macrophages in a glioblastoma rodent model. High level of VEGF led to threefold enlarged tumor volumes and a pronounced remodeling of the vascular structure along with a reduced infiltration of microglia/macrophages by approximately 50%. Remaining microglia/macrophages showed an enhanced rate of apoptosis as well as significant downregulation of the VEGF-receptor, VEGFR2, and others such as CXCR4. Consequently, we determined a substantially impaired migration of these microglia/macrophages to VEGF and SDF1α in vitro. Furthermore, we observed an increased presentation of the surface molecules MHCI and MHCII on microglia/macrophages from VEGF-overexpressing gliomas that are essential for activation of the adaptive immune system. In contrast, the expression of pro-inflammatory and suppressive cytokines, associated with the innate immune response, were mainly downregulated. Remarkably, the abundance of VEGF provoked less accumulation of microglia/macrophages within the perivascular niche and concomitantly reduced the release of pro-angiogenic factors, like VEGF, suggesting a possible regulatory feedback mechanism. Thus, the quantity of VEGF in the glioma microenvironment seems to be crucial for the participation of microglia/macrophages on tumor progression and should be considered for developing novel therapeutic approaches.

High miR-21 expression from FFPE tissues is associated with poor survival and response to adjuvant chemotherapy in colon cancer.

Colon cancer (CC) is a leading cause of cancer mortality. Novel biomarkers are needed to identify CC patients at high risk of recurrence and those who may benefit from therapeutic intervention. The aim of this study is to investigate if miR-21 expression from RNA isolated from formalin-fixed paraffin-embedded (FFPE) tissue sections is associated with prognosis and therapeutic outcome for patients with CC. The expression of miR-21 was measured by quantitative reverse transcriptase-polymerase chain reaction in a Japanese cohort (stage I-IV, n = 156) and a German cohort (stage II, n = 145). High miR-21 expression in tumors was associated with poor survival in both the stage II/III Japanese (p = 0.0008) and stage II German (p = 0.047) cohorts. These associations were independent of other clinical covariates in multivariable models. Receipt of adjuvant chemotherapy was not beneficial in patients with high miR-21 in either cohort. In the Japanese cohort, high miR-21 expression was significantly associated with poor therapeutic outcome (p = 0.0001) and adjuvant therapy was associated with improved survival in patients with low miR-21 (p = 0.001). These results suggest that miR-21 is a promising biomarker to identify patients with poor prognosis and can be accurately measured in FFPE tissues. The expression of miR-21 may also identify patients who will benefit from adjuvant chemotherapy.

The placenta protects the fetal circulation from anxiety-driven elevations in maternal serum levels of brain-derived neurotrophic factor.

Brain-derived neurotrophic factor (BDNF) plays crucial roles in brain function. Numerous studies report alterations in BDNF levels in human serum in various neurological conditions, including mood disorders such as depression. However, little is known about BDNF levels in the blood during pregnancy. We asked whether maternal depression and/or anxiety during pregnancy were associated with altered serum BDNF levels in mothers (n = 251) and their new-born infants (n = 212). As prenatal exposure to maternal mood disorders significantly increases the risk of neurological conditions in later life, we also examined the possibility of placental BDNF transfer by developing a new mouse model. We found no association between maternal symptoms of depression and either maternal or infant cord blood serum BDNF. However, maternal symptoms of anxiety correlated with significantly raised maternal serum BDNF exclusively in mothers of boys (r = 0.281; P = 0.005; n = 99). Serum BDNF was significantly lower in male infants than female infants but neither correlated with maternal anxiety symptoms. Consistent with this observation, we found no evidence for BDNF transfer across the placenta. We conclude that the placenta protects the developing fetus from maternal changes in serum BDNF that could otherwise have adverse consequences for fetal development.

Analysis of anti-proliferative and chemosensitizing effects of sunitinib on human esophagogastric cancer cells: Synergistic interaction with vandetanib via inhibition of multi-receptor tyrosine kinase pathways.

The receptor tyrosine kinases (RTKs), epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 1-3 (VEGFR1-3), are frequently expressed in gastric cancer and are putative therapeutic targets in this disease. We have investigated the anti-proliferative and chemosensitizing properties of the multitargeted small-molecule RTK inhibitors sunitinib and vandetanib in a panel of 4 human gastric and esophageal cancer cell lines. In the 1st instance, the expression of potential targets of these small-molecule inhibitors was examined by reverse transcriptase-polymerase chain reaction, western blotting, and flow cytometry. EGFR mRNA and protein was detected in all cases, with VEGFR2 expression noted in all but 1 line. Both EGF and VEGF were shown to stimulate tumor cell growth, and both sunitinib and vandetanib were found to be associated with significant dose-dependent inhibition of proliferation and an enhancement of apoptosis, as determined by MTT and propidium iodide/Annexin V labeling assays, respectively. The addition of sunitinib to VEGF-stimulated NCI-N87 cells was associated with a reduction in MAPK phosphorylation (pMAPK) but not Akt phosphorylation (pAkt), whereas the addition of vandetanib was associated with reductions in both VEGF- and EGF-mediated VEGFR2 phosphorylation, pMAPK and pAkt. Co-administration of sunitinib significantly enhanced the sensitivity of MKN-45 cells to cisplatin and irinotecan. In addition, vandetanib synergistically enhanced the sunitinib-associated inhibition of gastric cancer cell growth. In conclusion, these preliminary data confirm the importance of EGFR and VEGFR signaling in gastric cancer and suggest that the simultaneous inhibition of RTK-pathways through sunitinib and vandetanib may provide therapeutic benefit in this disease.

CCR2 of Tumor Microenvironmental Cells Is a Relevant Modulator of Glioma Biology.

Glioblastoma multiforme (GBM) shows a high influx of tumor-associated macrophages (TAMs). The CCR2/CCL2 pathway is considered a relevant signal for the recruitment of TAMs and has been suggested as a therapeutic target in malignant gliomas. We found that TAMs of human GBM specimens and of a syngeneic glioma model express CCR2 to varying extents. Using a Ccr2-deficient strain for glioma inoculation revealed a 30% reduction of TAMs intratumorally. This diminished immune cell infiltration occurred with augmented tumor volumes likely based on increased cell proliferation. Remaining TAMs in Ccr2-/- mice showed comparable surface marker expression patterns in comparison to wildtype mice, but expression levels of inflammatory transcription factors (Stat3, Irf7, Cox2) and cytokines (Ifnß, Il1ß, Il12α) were considerably affected. Furthermore, we demonstrated an impact on blood vessel integrity, while vascularization of tumors appeared similar between mouse strains. The higher stability and attenuated leakiness of the tumor vasculature imply improved sustenance of glioma tissue in Ccr2-/- mice. Additionally, despite TAMs residing in the perivascular niche in Ccr2-/- mice, their pro-angiogenic activity was reduced by the downregulation of Vegf. In conclusion, lacking CCR2 solely on tumor microenvironmental cells leads to enhanced tumor progression, whereby high numbers of TAMs infiltrate gliomas independently of the CCR2/CCL2 signal.

Interferon-γ signaling in human iPSC-derived neurons recapitulates neurodevelopmental disorder phenotypes.

Maternal immune activation increases the risk of neurodevelopmental disorders. Elevated cytokines, such as interferon-γ (IFN-γ), in offspring's brains play a central role. IFN-γ activates an antiviral cellular state, limiting viral entry and replication. Moreover, IFN-γ is implicated in brain development. We tested the hypothesis that IFN-γ signaling contributes to molecular and cellular phenotypes associated with neurodevelopmental disorders. Transient IFN-γ treatment of neural progenitors derived from human induced pluripotent stem cells increased neurite outgrowth. RNA sequencing analysis revealed that major histocompatibility complex class I (MHCI) genes were persistently up-regulated through neuronal differentiation-an effect that was mediated by IFN-γ-induced promyelocytic leukemia protein (PML) nuclear bodies. Critically, IFN-γ-induced neurite outgrowth required both PML and MHCI. We also found evidence that IFN-γ disproportionately altered the expression of genes associated with schizophrenia and autism, suggesting convergence between genetic and environmental risk factors. Together, these data implicate IFN-γ signaling in neurodevelopmental disorder etiology.

Using genome editing to engineer universal platelets.

Genome editing technologies such as zinc finger nucleases, TALENs and CRISPR/Cas9 have recently emerged as tools with the potential to revolutionise cellular therapy. This is particularly exciting for the field of regenerative medicine, where the large-scale, quality-controlled editing of large numbers of cells could generate essential cellular products ready to move towards the clinic. This review details recent progress towards generating HLA Class I null platelets using genome editing technologies for ß2-microglobulin deletion, generating a universally transfusable cellular product. In addition, we discuss various methods for megakaryocyte (MK) production from human pluripotent stem cells and subsequent platelet production from the MKs. As well as simply producing platelets, differentiating MK cultures can enable us to understand megakaryopoiesis in vivo and take steps towards ameliorating bleeding disorders or deficiencies in MK maturation in patients. Thus by intersecting both these areas of research, we can produce optimised differentiation systems for the production of universal platelets, thus offering a stable supply of platelets for difficult-to-match patients and providing areas with transmissible disease concerns or an unpredictable supply of platelets with a steady supply of quality-controlled platelet units.

VEGF-D expression correlates with colorectal cancer aggressiveness and is downregulated by cetuximab.

AIM: To gain mechanistic insights into the role played by epidermal growth factor receptor (EGFR) in the regulation of vascular endothelial growth factors (VEGFs) in colorectal cancer (CRC). METHODS: The impact of high-level expression of the growth factor receptors EGFR and VEGF receptor (VEGFR)3 and the VEGFR3 ligands VEGF-C and VEGF-D on disease progression and prognosis in human CRC was investigated in 108 patients using immunohistochemistry. Furthermore, the expression of the lymphangiogenic factors in response to the modulation of EGFR signalling by the EGFR-targeted monoclonal antibody cetuximab was investigated at the mRNA and protein level in human SW480 and SW620 CRC cell lines and a mouse xenograft model. RESULTS: Human CRC specimens and cell lines displayed EGFR, VEGF-C and VEGF-D expression with varying intensities. VEGF-C expression was associated with histological grade. Strong expression of VEGF-D was significantly associated with lymph node metastases and linked to a trend for decreased survival in lymph node-positive patients. EGFR blockade with cetuximab resulted in a significant decrease of VEGF-D expression in vitro and in vivo. CONCLUSION: In conclusion, the expression of VEGF-D in colorectal tumours is significantly associated with lymphatic involvement in CRC patients and such expression might be blocked effectively by cetuximab.

Transcription Factor Levels after Forward Programming of Human Pluripotent Stem Cells with GATA1, FLI1, and TAL1 Determine Megakaryocyte versus Erythroid Cell Fate Decision.

The production of blood cells and their precursors from human pluripotent stem cells (hPSCs) in vitro has the potential to make a significant impact upon healthcare provision. We demonstrate that the forward programming of hPSCs through overexpression of GATA1, FLI1, and TAL1 leads to the production of a population of progenitors that can differentiate into megakaryocyte or erythroblasts. Using "rainbow" lentiviral vectors to quantify individual transgene expression in single cells, we demonstrate that the cell fate decision toward an erythroblast or megakaryocyte is dictated by the level of FLI1 expression and is independent of culture conditions. Early FLI1 expression is critical to confer proliferative potential to programmed cells while its subsequent silencing or maintenance dictates an erythroid or megakaryocytic fate, respectively. These committed progenitors subsequently expand and mature into megakaryocytes or erythroblasts in response to thrombopoietin or erythropoietin. Our results reveal molecular mechanisms underlying hPSC forward programming and novel opportunities for application to transfusion medicine.

Structurally graduated collagen scaffolds applied to the ex vivo generation of platelets from human pluripotent stem cell-derived megakaryocytes: Enhancing production and purity.

Platelet transfusions are a key treatment option for a range of life threatening conditions including cancer, chemotherapy and surgery. Efficient ex vivo systems to generate donor independent platelets in clinically relevant numbers could provide a useful substitute. Large quantities of megakaryocytes (MKs) can be produced from human pluripotent stem cells, but in 2D culture the ratio of platelets harvested from MK cells has been limited and restricts production rate. The development of biomaterial cell supports that replicate vital hematopoietic micro-environment cues are one strategy that may increase in vitro platelet production rates from iPS derived Megakaryocyte cells. In this paper, we present the results obtained generating, simulating and using a novel structurally-graded collagen scaffold within a flow bioreactor system seeded with programmed stem cells. Theoretical analysis of porosity using micro-computed tomography analysis and synthetic micro-particle filtration provided a predictive tool to tailor cell distribution throughout the material. When used with MK programmed stem cells the graded scaffolds influenced cell location while maintaining the ability to continuously release metabolically active CD41 + CD42 + functional platelets. This scaffold design and novel fabrication technique offers a significant advance in understanding the influence of scaffold architectures on cell seeding, retention and platelet production.

Myeloid cells expressing high level of CD45 are associated with a distinct activated phenotype in glioma.

Glioblastoma multiforme is characterized by high accumulation of microglia/macrophages. The function of these tumor-infiltrating myeloid cells is not sufficiently elucidated. Therefore, a better understanding of the precise immune cell composition and function in brain tumors is required. In rodent glioma models, two different myeloid cell populations exist, determined by the expression level of CD45, namely CD11b+CD45low and CD11b+CD45high. Previous analyses of cytokine and marker expression profiles were almost exclusively performed on the entire myeloid cell fraction. Consequently, described pro- and anti-tumoral characteristics were not assigned to the evident subpopulations. In the present study, we used a syngeneic glioblastoma mouse model and subsequent flow cytometric analyses to demonstrate the distinct properties of CD11b+CD45high and the CD11b+CD45low cells. First, the majority of CD11b+CD45high cells expressed high level of GR1 and around 6% of IL10 representing in part features of myeloid-derived suppressor cells, while the CD11b+CD45low fraction displayed no upregulation of these molecules. Second, we detected that specifically the CD11b+CD45high population showed antigen-presenting, co-stimulatory, and inflammatory features. Here, we identified up to 80% of MHCII and approximately 50% of CD86 and TNFα-expressing cells. Investigation of MHCI and CD80 revealed a moderate upregulation. By contrast, in the CD11b+CD45low cell fraction, merely MHCII and TNFα were marginally overexpressed. In summary, these data emphasize the specific phenotype of CD11b+CD45high cells in glioma with suppressive as well as pro-inflammatory characteristics whereas the CD11b+CD45low cells were almost unaffected. Hence, primarily, the subpopulation consisting of CD45high-expressing cells is activated by the tumor and should be considered as therapeutic target.

Selective PI3K inhibition by BKM120 and BEZ235 alone or in combination with chemotherapy in wild-type and mutated human gastrointestinal cancer cell lines.

PURPOSE: New targeted agents like antibodies or small molecules against tyrosine and lipid kinases clearly expand the standard therapy options in oncology. However, tumour resistance is still a challenge, often induced by mutations in growth-related signalling cascades. Twenty and ten percentage of all patients with colorectal and gastric cancers, respectively, carry phosphatidyl-3-kinase (PI3K) mutations and do not respond to receptor-blocking therapies. Recently, selective kinase inhibitors have been generated, which block the PI3K signalling pathway in tumour cells. So far, their therapeutic role for the treatment of mutated versus wild-type human gastrointestinal cancers has not been clarified in detail. METHODS: To define the inhibitory and pro-apoptotic effects of the two PI3K inhibitors BEZ235 and BKM120 in three human colon cancer (HT-29, HCT-116 and DLD-1) and three gastric cancer (NCI-n87, AGS and MKN-45), cell lines with different PIK3CA gene mutation status were used. Firstly, viability, apoptosis and caspase assays were performed during incubation with either the inhibitors alone or combined with different cytotoxic agents. Secondly, the molecular consequences for the cell cycle and signalling pathways were analysed by defining the protein levels by FACS and Western blot analysis. RESULTS: Both the PI3K inhibitors BEZ235 and BKM120 induced a clear concentration-dependent reduction in cell viability and an increase in apoptotic cell death, with the mutated cells being more sensitive to treatment. However, single-agent BEZ235 caused a G1 arrest in tumour cells, whilst BKM120 induced a G2 shift in a half of the gastrointestinal cancer cell lines. There was a clear downregulation in the protein levels of the PI3K-AKT pathway at the concentrations of 100 nM for both agents and for BEZ235 the additional inhibition of the mTOR pathway. Furthermore, BEZ235 caused synergistic induction of apoptosis when combined with irinotecan in colon cancer cell lines. Human gastric cancer cells were less sensitive to both BEZ235 and BKM120. CONCLUSIONS: BEZ235 and BKM120 induced pro-apoptotic effects in all cell lines and especially with an increased response in the PI3KCA mutated cells. Our data support the clinical development of these PI3K inhibitors for patients with wild-type or mutated colon cancers.