CFU-S(11) activity does not localize solely with the aorta in the aorta-gonad-mesonephros region

The aorta-gonad-mesonephros (AGM) region is a potent hematopoietic site in the midgestation mouse conceptus and first contains colony-forming units-spleen day 11 (CFU-S(11)) at embryonic day 10 (E10). Because CFU-S(11) activity is present in the AGM region before the onset of hematopoietic stem cell (HSC) activity, CFU-S(11) activity in the complex developing vascular and urogenital regions of the AGM was localized. From E10 onward, CFU-S(11) activity is associated with the aortic vasculature, and is found also in the urogenital ridges (UGRs). Together with data obtained from organ explant cultures, in which up to a 16-fold increase in CFU-S(11) activity was observed, it was determined that CFU-S(11) can be increased autonomously both in vascular sites and in UGRs. Furthermore, CFU-S(11) activity is present in vitelline and umbilical vessels. This, together with the presence of CFU-S(11) in the UGRs 2 days before HSC activity, suggests both temporally and spatially distinct emergent sources of CFU-S(11). (Blood. 2000;96:2902-2904

Allogeneic chondrogenically differentiated human bone marrow stromal cells do not induce dendritic cell maturation

Bone marrow stromal cell (BMSC)-mediated endochondral bone formation may be a promising alternative to the current gold standards of autologous bone transplantation, in the development of novel methods for bone repair. Implantation of chondrogenically differentiated BMSCs leads to bone formation in vivo via endochondral ossification. The success of this bone formation in an allogeneic system depends upon the interaction between the implanted constructs and the host immune system. The current study investigated the effect of chondrogenically differentiated human bone marrow stromal cell (hBMSC) pellets on the maturation and function of dendritic cells (DCs) by directly coculturing bone forming chondrogenic hBMSC pellets and immature or lipopolysaccharide (LPS)-matured DCs in vitro. Allogeneic chondrogenic hBMSC pellets did not affect the expression of CD80, CD86, or HLADR on immature or LPS-matured DCs following 24, 48, or 72 hr of coculture. Furthermore, they did not induce or inhibit antigen uptake or migration of the DCs over time. IL-6 was secreted by allogeneic chondrogenic hBMSC pellets in response to LPS-matured DCs. Overall, this study has demonstrated that maturation of immature DCs was not influenced by allogeneic chondrogenic hBMSC pellets. This suggests that allogeneic chondrogenic hBMSC pellets do not stimulate immunogenic responses from DCs in vitro and are not expected to indirectly activate T cells via DCs. For this reason, allogeneic chondrogenic bone marrow stromal cell pellets are promising candidates for future tissue engineering strategies utilising allogeneic cells for bone repair

PDX1 DNA methylation distinguishes two subtypes of pancreatic neuroendocrine neoplasms with a different prognosis

DNA methylation is a crucial epigenetic mechanism for gene expression regulation and cell differentiation. Furthermore, it was found to play a major role in multiple pathological processes, including cancer. In pancreatic neuroendocrine neoplasms (PNENs), epigenetic deregulation is also considered to be of significance, as the most frequently mutated genes have an important function in epigenetic regulation. However, the exact changes in DNA methylation between PNENs and the endocrine cells of the pancreas, their likely cell-of-origin, remain largely unknown. Recently, two subtypes of PNENs have been described which were linked to cell-of-origin and have a different prognosis. A difference in the expression of the transcription factor PDX1 was one of the key molecular differences. In this study, we performed an exploratory genome-wide DNA methylation analysis using Infinium Methylation EPIC arrays (Illumina) on 26 PNENs and pancreatic islets of five healthy donors. In addition, the methylation profile of the PDX1 region was used to perform subtyping in a global cohort of 83 PNEN, 2 healthy alpha cell and 3 healthy beta cell samples. In our exploratory analysis, we identified 26,759 differentially methylated CpGs and 79 differentially methylated regions. The gene set enrichment analysis highlighted several interesting pathways targeted by altered DNA methylation, including MAPK, platelet-related and immune system-related pathways. Using the PDX1 methylation in 83 PNEN, 2 healthy alpha cell and 3 healthy beta cell samples, two subtypes were identified, subtypes A and B, which were similar to alpha and beta cells, respectively. These subtypes had different clinicopathological characteristics, a different pattern of chromosomal alterations and a different prognosis, with subtype A having a significantly worse prognosis compared with subtype B (HR 0.22 [95% CI: 0.051–0.95], p = 0.043). Hence, this study demonstrates that several cancer-related pathways are differently methylated between PNENs and normal islet cells. In addition, we validated the use of the PDX1 methylation status for the subtyping of PNENs and its prognostic importance

Semiautomated isolation and molecular characterisation of single or highly purified tumour cells from CellSearch enriched blood samples using dielectrophoretic cell sorting

Background: Molecular characterisation of single circulating tumour cells (CTCs) holds considerable promise for predictive biomarker assessment and to explore CTC heterogeneity. We evaluate a new method, the DEPArray system, that allows the dielectrophoretic manipulation and isolation of single and 100% purified groups of CTCs from pre-enriched blood samples and explore the feasibility of their molecular characterisation.Methods:Samples containing known numbers of two cell populations were used to assess cell loss during sample loading. Cultured breast cancer cells were isolated from spiked blood samples using CellSearch CTC and Profile kits. Single tumour cells and groups of up to 10 tumour cells were recovered with the DEPArray system and subjected to transcriptional and mutation analysis.Results:On average, 40% cell loss was observed when loading samples to the DEPArray system. Expected mutations in clinically relevant markers could be obtained for 60% of single recovered tumour cells and all groups of tumour cells. Reliable gene expression profiles were obtained from single cells and groups of up to 10 cells for 2 out of 3 spiked breast cancer cell lines.Conclusion:We describe a semiautomated workflow for the isolation of small groups of 1 to 10 tumour cells from whole blood samples and provide proof of principle for the feasibility of their comprehensive molecular characterisation

Long-term acquired everolimus resistance in pancreatic neuroendocrine tumours can be overcome with novel PI3K-AKT-mTOR inhibitors

Background:The mTOR-inhibitor everolimus improves progression-free survival in advanced pancreatic neuroendocrine tumours (PNETs). However, adaptive resistance to mTOR inhibition is described.Methods:QGP-1 and BON-1, two human PNET cell lines, were cultured with increasing concentrations of everolimus up to 22 weeks to reach a dose of 1 μM everolimus, respectively, 1000-fold and 250-fold initial IC 50. Using total DNA content as a measure of cell number, growth inhibitory dose-response curves of everolimus were determined at the end of resistance induction and over time after everolimus withdrawal. Response to ATP-competitive mTOR inhibitors OSI-027 and AZD2014, and PI3K-mTOR inhibitor NVP-BEZ235 was studied. Gene expression of 10 PI3K-Akt-mTOR pathway-related genes was evaluated using quantitative real-time PCR (RT-qPCR).Results:Long-term everolimus-treated BON-1/R and QGP-1/R showed a significant reduction in everolimus sensitivity. During a drug holiday, gradual return of everolimus sensitivity in BON-1/R and QGP-1/R led to complete reversal of resistance after 10-12 weeks. Treatment with AZD2014, OSI-027 and NVP-BEZ235 had an inhibitory effect on cell proliferation in both sensitive and resistant cell lines. Gene expression in BON-1/R revealed downregulation of MTOR, RICTOR, RAPTOR, AKT and HIF1A, whereas 4EBP1 was upregulated. In QGP-1/R, a downregulation of HIF1A and an upregulation of ERK2 were observed.Conclusions:Long-term everolimus resistance was induced in two human PNET cell lines. Novel PI3K-AKT-mTOR pathway-targeting drugs can overcome everolimus resistance. Differential gene expression profiles suggest different mechanisms of everolimus resistance in BON-1 and QGP-1

HER-2 status of circulating tumor cells in a metastatic breast cancer cohort: A comparative study on characterization techniques

Background Personalized targeted treatment in metastatic breast cancer relies on accurate assessment of molecular aberrations, e.g. overexpression of Human Epidermal growth factor Receptor 2 (HER-2). Molecular interrogation of circulating tumor cells (CTCs) can provide an attractive alternative for real-time biomarker assessment. However, implementation of CellSearch-based HER-2 analysis has been limited. Immunofluorescent (IF) image interpretation is crucial, as different HER-2 categories have been described. Major questions in CTC research are how these IF categories reflect gene expression and amplification, and if we should consider ‘medium’ HER-2 expressing CTCs for patient selection. Methods Tumor cells from spiked cell lines (n = 8) and CTCs (n = 116 samples) of 85 metastatic breast cancer patients were enriched using CellSearch. Comparative analysis of HER-2 expression by IF imaging (ACCEPT, DEPArray, and visual scoring) with qRT-PCR and HER-2/neu FISH was performed. Results Automated IF HER-2-profiling by DEPArray and ACCEPT delivered comparable results. There was a 98% agreement between 17 trained observers (visual scoring) and ACCEPT considering HER-2neg and HER-2high expressing CTCs. However, 89% of HER-2med expressing CTCs by ACCEPT were scored negative by observers. HER-2high expressing tumor cells demonstrated HER-2/neu gene amplification, whereas HER-2neg and HER-2med expressing tumor cells and CTCs by ACCEPT were copy-number neutral. All patients with HER-2-positive archival tumors had �1 HER-2high expressing CTCs, while 80% of HER-2- negative patients did not. High relative gene expression of HER-2 measured on enriched CTC lysates correlated with having �1 HER-2high expressing CTCs. Conclusion Automated images analysis has enormous potential for clinical implementation. HER-2 characterization and clinical trial design should be focused on HER-2high expressing CTCs

Circulating cell-free nucleosomes as biomarker for kidney transplant rejection: a pilot study

Background: There is an unmet need for noninvasive markers specific for kidney transplant rejection. Such a marker may eventually overcome the need for a transplant biopsy. In this pilot study, the potential of circulating cell-free nucleosomes (CCFN) to serve as a biomarker for kidney transplant rejection was evaluated. Methods: Forty de novo kidney transplant recipients were prospectively followed as part of a randomized, controlled clinical trial. Total CCFN (H3) and CCFN with the histone modifications H3K36me3 and H3 citrulline were measured in patients at four fixed time points: before transplantation and on days 3–6, 30 and 180 after kidney transplantation. In addition, serum collected at times of transplant rejection (n = 14) was analyzed. CCFN were measured with a Nu.Q™ Assay kit (VolitionRx), an ELISA-based assay using antibodies directed against nucleosomes. Results: For total CCFN (H3), H3K36me3, and H3 citrulline, the same pattern was seen over time: Concentrations were elevated shortly after transplantation (day 3–6) followed by a decline reaching baseline (pre-transplantation) values at days 30 and 180. At times of acute rejection, the median concentration of total CCFN (H3) was significantly higher compared to the stable situation (day 30): 4309 (3435–5285) versus 2885 (1668–3923) ng/mL, p < 0.05, respectivel

Elevated Expression of Phospholipid Transfer Protein in Bone Marrow Derived Cells Causes Atherosclerosis

Background: Phospholipid transfer protein (PLTP) is expressed by various cell types. In plasma, it is associated with high density lipoproteins (HDL). Elevated levels of PLTP in transgenic mice result in decreased HDL and increased atherosclerosis. PLTP is present in human atherosclerosis lesions, where it seems to be macrophage derived. The aim of the present study is to evaluate the atherogenic potential of macrophage derived PLTP. Methods and Findings: Here we show that macrophages from human PLTP transgenic mice secrete active PLTP. Subsequently, we performed bone marrow transplantations using either wild type mice (PLTPwt/wt), hemizygous PLTP transgenic mice (huPLTPtg/wt) or homozygous PLTP transgenic mice (huPLTPtg/tg) as donors and low density lipoprotein receptor deficient mice (LDLR-/-) as acceptors, in order to establish the role of PLTP expressed by bone marrow derived cells in diet-induced atherogenesis. Atherosclerosis was increased in the huPLTPtg/wt → LDLR-/ - mice (2.3-fold) and even further in the huPLTPtg/tg→LDLR-/ - mice (4.5-fold) compared with the control PLTPwt/wt→LDLR-/- mice (both P<0.001). Plasma PLTP activity levels and non-HDL cholesterol were increased and HDL cholesterol decreased compared with controls (all P<0.01). PLTP was present in atherosclerotic plaques in the mice as demonstrated by immunohistochemistry and appears to co-localize with macrophages. Isolated macrophages from PLTP transgenic mice do not show differences in cholesterol efflux or in cytokine production. Lipopolysaccharide activation of macrophages results in increased production of PLTP. This effect was strongly amplified in PLTP transgenic macrophages. Conclusions: We conclude that PLTP expression by bone marrow derived cells results in atherogenic effects on plasma lipids, increased PLTP activity, high local PLTP protein levels in the atherosclerotic lesions and increased atherosclerotic lesion size