Nanocytometer for smart analysis of peripheral blood and acute myeloid leukemia: a pilot study.

We realize an ultracompact nanocytometer for real-time impedimetric detection and classification of subpopulations of living cells. Nanoscopic nanowires in a microfluidic channel act as nanocapacitors and measure in real time the change of the amplitude and phase of the output voltage and, thus, the electrical properties of living cells. We perform the cell classification in the human peripheral blood (PBMC) and demonstrate for the first time the possibility to discriminate monocytes and subpopulations of lymphocytes in a label-free format. Further, we demonstrate that the PBMC of acute myeloid leukemia and healthy samples grant the label free identification of the disease. Using the algorithm based on machine learning, we generated specific data patterns to discriminate healthy donors and leukemia patients. Such a solution has the potential to improve the traditional diagnostics approaches with respect to the overall cost and time effort, in a label-free format, and restrictions of the complex data analysis.

Androgens induce a distinct response of epithelial-mesenchymal transition factors in human prostate cancer cells.

Inhibition of the androgen receptor (AR) is a major target of prostate cancer (PCa) therapy. However, prolonged androgen deprivation results eventually in castration-resistant PCa (CRPC) with metastasis and poor survival. Emerging evidence suggests that epithelial-mesenchymal transition (EMT) may facilitate castration-resistance and cancer metastasis in PCa. The human androgen-dependent, castration-sensitive prostate cancer (CSPC) cell line LNCaP and the CRPC cell line C4-2 are often used as a model system for human PCa. However, the role of the AR and the effect of AR antagonist (antiandrogen) treatment on the RNA expression of key factors of EMT including the long non-coding RNAs (lncRNAs) DRAIC in PCa cells remain elusive. Although as expected the established AR target genes PSA and FKBP5 are strongly induced by androgens in both cell lines, both E-cadherin and vimentin mRNA levels are upregulated by androgens in LNCaP but not in C4-2 cells by short- and long-term treatments. The mRNA levels of E-cadherin and vimentin remain unchanged by antiandrogen treatment in both cell lines. The expression of transcription factors that regulate EMT including Slug, Snail and ZEB1 and the lncRNA DRAIC were affected by androgen treatment in both cell lines. The mRNA level of Slug is upregulated by androgens and interestingly downregulated by antiandrogens in both cell lines. On the other hand, ZEB1 mRNA levels are strongly upregulated by androgens but remain unchanged by antiandrogens. In contrast, Snail mRNA levels are repressed by androgen treatment similar to DRAIC RNA levels. However, while antiandrogen treatment seems not to change Snail mRNA levels, antiandrogen treatments induce DRAIC RNA levels. Moreover, despite the strong upregulation of Zeb1 mRNA, no significant increase of the ZEB1 protein was observed indicating that despite androgen upregulation, posttranscriptional regulation of EMT controlling transcription factors occurs. SLUG protein was enhanced in both cell lines by androgens and reduced by antiandrogens. Taken together, our data suggest that the ligand-activated AR regulates the expression of several EMT key factors and antiandrogens counteract AR activity only on selected genes.

Role of osteogenic Dickkopf-1 in bone remodeling and bone healing in mice with type I diabetes mellitus.

Type 1 diabetes mellitus (T1DM) is associated with low bone mass and a higher risk for fractures. Dickkopf-1 (Dkk1), which inhibits Wnt signaling, osteoblast function, and bone formation, has been found to be increased in the serum of patients with T1DM. Here, we investigated the functional role of Dkk1 in T1DM-induced bone loss in mice. T1DM was induced in 10-week-old male mice with Dkk1-deficiency in late osteoblasts/osteocytes (Dkk1f/f;Dmp1-Cre, cKO) and littermate control mice by 5 subsequent injections of streptozotocin (40 mg/kg). Age-matched, non-diabetic control groups received citrate buffer instead. At week 12, calvarial defects were created in subgroups of each cohort. After a total of 16 weeks, weight, fat, the femoral bone phenotype and the area of the bone defect were analyzed using µCT and dynamic histomorphometry. During the experiment, diabetic WT and cKO mice did not gain body weight compared to control mice. Further they lost their perigonadal and subcutaneous fat pads. Diabetic mice had highly elevated serum glucose levels and impaired glucose tolerance, regardless of their Dkk1 levels. T1DM led to a 36% decrease in trabecular bone volume in Cre- negative control animals, whereas Dkk1 cKO mice only lost 16%. Of note, Dkk1 cKO mice were completely protected from T1DM-induced cortical bone loss. T1DM suppressed the bone formation rate, the number of osteoblasts at trabecular bone, serum levels of P1NP and bone defect healing in both, Dkk1-deficient and sufficient, mice. This may be explained by increased serum sclerostin levels in both genotypes and the strict dependence on bone formation for bone defect healing. In contrast, the number of osteoclasts and TRACP 5b serum levels only increased in diabetic control mice, but not in Dkk1 cKO mice. In summary, Dkk1 derived from osteogenic cells does not influence the development of T1DM but plays a crucial role in T1DM-induced bone loss in male mice by regulating osteoclast numbers.

Contributions of Dickkopf-1 to Obesity-Induced Bone Loss and Marrow Adiposity.

Low bone strength in overweight individuals is a significant medical problem. One important determinant of mesenchymal stem cell fate into osteoblasts or adipocytes is the Wnt signaling pathway. We recently showed that Dickkopf-1 (DKK1), a potent Wnt inhibitor, is upregulated in obese mice. In this study, we investigated the role of DKK1 in the pathogenesis of obesity-induced bone loss using global and tissue-specific KO mice. Obesity was induced in 8-week-old male mice with an inducible global (Rosa26-CreERT2) or osteoprogenitor- (Osx-Cre-) specific deletion of Dkk1 with a high-fat diet (HFD) containing 60% fat. After 12 weeks, body weight, bone volume, bone fat mass, and bone turnover were assessed. Dkk1 fl/fl ;Rosa26-CreERT2 mice experienced a similar increase in body weight and white fat pads as control mice. A HFD significantly reduced trabecular bone mass and the bone formation rate in Cre- mice and Dkk1 fl/fl ;Rosa26-CreERT2 mice. Interestingly, Dkk1 fl/fl ;Rosa26-CreERT2 mice were protected from HFD-induced cortical bone loss. Furthermore, a HFD was associated with increased bone marrow fat in the femur, which was less pronounced in Dkk1 fl/fl ;Rosa26-CreERT2 mice. Mice with an osteoprogenitor-specific Dkk1 deletion showed similar results as the global knockout, showing a protection against HFD-induced cortical bone loss and an accumulation of bone marrow fat, but a similar decrease in trabecular bone volume. In summary, DKK1 appears to contribute distinctly to cortical, but not trabecular bone loss in obesity. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Osteogenic Dkk1 Mediates Glucocorticoid-Induced but Not Arthritis-Induced Bone Loss.

Dickkopf-1 (Dkk1) is a negative regulator of bone formation and bone mass and is deregulated in bone loss induced by arthritis and glucocorticoid (GC) exposure. However, the role of Dkk1 in these pathological processes is still unknown. Here, we used conditional Dkk1 knock-out mice to determine the role of Dkk1 produced by osteolineage cells in the development of arthritis and GC-induced bone loss. Osteoprogenitor (Osx-Cre)- and osteocyte (Dmp1-Cre)-specific knock-out mice and their Cre-negative controls were subjected to two arthritis models, K/BxN and antigen-induced arthritis. Disease induction and progression were assessed. GC-induced bone loss was induced in 25-week-old female mice by implanting prednisolone (7.5 mg) slow-release pellets for 4 weeks. Dkk1fl/fl ;Osx-Cre mice subjected to K/BxN arthritis showed mildly reduced disease severity with reduced infiltration of neutrophils and T cells into affected joints and reduced bone erosions compared with Cre-negative controls. Osteocyte-specific Dkk1 deletion did not affect disease severity or local bone erosions. However, systemic bone loss at the spine was less severe in both mouse lines. In contrast to arthritis, both lines were protected from GC-induced bone loss. Although the Cre-negative controls lost about 26% and 31% bone volume potentially caused by decreased bone formation, Cre-positive mice did not exhibit such alterations. Dkk-1 deficiency in osteolineage cells protects against GC-induced bone loss, whereas it had only minor effects in arthritis. Therefore, Dkk1 may be a promising therapeutic target especially for bone diseases in which inhibition of bone formation represents the predominant mechanism. © 2019 American Society for Bone and Mineral Research.

The Role of Dickkopf-1 in Thyroid Hormone-Induced Changes of Bone Remodeling in Male Mice.

Thyroid hormones regulate bone homeostasis, and exogenously induced hyperthyroidism and hypothyroidism in mice was recently found to be associated with an altered expression of the Wnt inhibitor Dickkopf-1 (Dkk1), a determinant of bone mass. Here, we assessed the role of Dkk1 in thyroid hormone-induced changes in bone using conditional Dkk1 knockout mice. Male mice with a global (Dkk1fl/fl;Rosa26-CreERT2) or osteocyte-specific (Dkk1fl/fl;Dmp1:Cre) deletion of Dkk1 were pharmacologically rendered hypothyroid or hyperthyroid. The bone phenotype was analyzed using micro-CT analysis, dynamic histomorphometry, and serum concentrations of bone turnover markers. Hypothyroid and hyperthyroid Cre-negative mice of either Cre line revealed the expected changes in bone volume with hypothyroid mice displaying a 40% to 60% increase in vertebral trabecular bone volume, while hyperthyroid mice lost 45% to 60% of bone volume. Similar changes were observed at the spine. Interestingly, Cre-positive mice of both lines did not gain or lose as much bone at the femur when rendered hypothyroid or hyperthyroid. While Cre-negative hypothyroid mice gained 80% to 100% bone volume, Cre-positive hypothyroid mice only increased their bone volume by 55% to 90%. Similarly, Cre-negative hyperthyroid mice lost 74% to 79% bone, while Cre-positive hyperthyroid mice merely lost 40% to 54%. Despite these site-specific differences, both global and osteocyte-specific Dkk1 knockout mice displayed similar changes in bone turnover as their Cre-negative controls in the hypothyroid and hyperthyroid states. While osteoblast and osteoclast parameters were increased in hyperthyroidism, hypothyroidism potently suppressed bone cell activities. Loss of Dkk1 is not sufficient to fully reverse thyroid hormone-induced changes in bone mass and bone turnover.

Postnatal Skeletal Deletion of Dickkopf-1 Increases Bone Formation and Bone Volume in Male and Female Mice, Despite Increased Sclerostin Expression.

The Wnt antagonist Dickkopf-1 (Dkk1) is a negative regulator of osteoblast function and bone mass. However, because of the lack of appropriate models, many aspects of its role in the regulation of postnatal bone turnover and its cellular source have remained unknown. In this study, we deleted Dkk1 postnatally and in different cell types using various Cre-drivers (Rosa26-ERT2-Cre, Osx-cre, Dmp1-Cre) and assessed to which extent cells of the osteoblastic lineage contribute to the effects of Dkk1 on bone turnover and homeostasis. Female and male mice were examined at 12 weeks of age. Mice with a global or cell type-specific deletion of Dkk1 showed a two- to threefold higher bone volume compared with their Cre-negative littermates. The mineral apposition rate and the bone formation rate were increased two- to fourfold in all three mouse lines, despite a significant increase in systemic and skeletal levels of sclerostin. Dkk1 deletion further reduced the number of osteoclasts about twofold, which was accompanied by a strong decrease in the receptor activator of nuclear factor-κB ligand/osteoprotegerin mRNA ratio in femoral bone. Despite similar increases in bone mass, the deletion of Dkk1 in osterix-expressing cells reduced circulating Dkk1 significantly (males, -79%; females, -77%), whereas they were not changed in Dkk1fl/fl ;Dmp1-Cre mice. However, both lines showed significantly reduced Dkk1 mRNA levels in bone. In summary, we show that lack of Dkk1 in cells of the osteoblastic lineage leads to high bone mass with increased bone formation, despite increased levels of sclerostin. Moreover, the majority of systemic Dkk1 appears to originate from osteoprogenitors but not from mature osteoblasts or osteocytes. Nevertheless, the amount of Dkk1 produced locally by more mature osteogenic cells is sufficient to modulate bone mass. Thus, this study highlights the importance of local Wnt signaling on postnatal bone homeostasis. © 2018 American Society for Bone and Mineral Research.