Ex Vivo Model of Neuroblastoma Plasticity

Simple Summary The complexity of tumor cell plasticity is still poorly understood. In particular, cellular changes during the metastatic process are difficult to monitor. This is a descriptive study of cell lines derived from primary tumors of xenografted LAN-1 cells and the corresponding three generations of bone metastases. Our results of ex vivo analysis of the cell lines depict the ability of tumor cells to adapt and survive in different microenvironments undergoing significant cellular alterations. The cell lines show strong phenotypical and biochemical changes and even an altered response to immune cells and chemotherapy. In conclusion, this mouse model allows to analyze the complex changes in tumor cell populations during metastasis and can be adapted to cell lines from different tumor origins. Abstract Tumor plasticity is essential for adaptation to changing environmental conditions, in particular during the process of metastasis. In this study, we compared morphological and biochemical differences between LAN-1 neuroblastoma (NB) cells recovered from a subcutaneous xenograft primary tumor (PT) and the corresponding three generations of bone metastasis (BM I–III). Moreover, growth behavior, as well as the response to chemotherapy and immune cells were assessed. For this purpose, F-actin was stained, mRNA and protein expression assessed, and lactate secretion analyzed. Further, we measured adhesion to collagen I, the growth rate of spheroids in the presence and absence of vincristine, and the production of IL-6 by peripheral blood mononuclear cells (PBMCs) co-incubated with PT or BM I–III. Analysis of PT and the three BM generations revealed that their growth rate decreased from PT to BM III, and accordingly, PT cells reacted most sensitively to vincristine. In addition, morphology, adaption to hypoxic conditions, as well as transcriptomes showed strong differences between the cell lines. Moreover, BM I and BM II cells exhibited a significantly different ability to stimulate human immune cells compared to PT and BM III cells. Interestingly, the differences in immune cell stimulation corresponded to the expression level of the cancer-testis antigen MAGE-A3. In conclusion, our ex vivo model allows to analyze the adaption of tumor populations to different microenvironments and clearly demonstrates the strong alteration of tumor cell populations during this process

ARSB IS REQUIRED FOR BONE REMODELING

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Conditional mouse models support the role of SLC39A14 (ZIP14) in Hyperostosis Cranialis Interna and in bone homeostasis

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An additional Lrp4 high bone mass mutation mitigates the sost-knockout phenotype in mice by increasing bone remodeling

Abstract: Pathogenic variants disrupting the binding between sclerostin (encoded by SOST) and its receptor LRP4 have previously been described to cause sclerosteosis, a rare high bone mass disorder. The sclerostin-LRP4 complex inhibits canonical WNT signaling, a key pathway regulating osteoblastic bone formation and a promising therapeutic target for common bone disorders, such as osteoporosis. In the current study, we crossed mice deficient for Sost (Sost(-/-)) with our p.Arg1170Gln Lrp4 knock-in (Lrp4(KI/KI)) mouse model to create double mutant Sost(-/-);Lrp4(KI/KI) mice. We compared the phenotype of Sost(-/-) mice with that of Sost(-/-);Lrp4(KI/KI) mice, to investigate a possible synergistic effect of the disease-causing p.Arg1170Trp variant in Lrp4 on Sost deficiency. Interestingly, presence of Lrp4(KI) alleles partially mitigated the Sost(-/-) phenotype. Cellular and dynamic histomorphometry did not reveal mechanistic insights into the observed phenotypic differences. We therefore determined the molecular effect of the Lrp4(KI) allele by performing bulk RNA sequencing on Lrp4(KI/KI) primary osteoblasts. Unexpectedly, mostly genes related to bone resorption or remodeling (Acp5, Rankl, Mmp9) were upregulated in Lrp4(KI/KI) primary osteoblasts. Verification of these markers in Lrp4(KI/KI), Sost(-/-) and Sost(-/-);Lrp4(KI/KI) mice revealed that sclerostin deficiency counteracts this Lrp4(KI/KI) effect in Sost(-/-);Lrp4(KI/KI) mice. We therefore hypothesize that models with two inactivating Lrp4(KI) alleles rather activate bone remodeling, with a net gain in bone mass, whereas sclerostin deficiency has more robust anabolic effects on bone formation. Moreover, these effects of sclerostin and Lrp4 are stronger in female mice, contributing to a more severe phenotype than in males and more detectable phenotypic differences among different genotypes

Gnathodiaphyseal dysplasia is not recapitulated in a respective mouse model carrying a mutation of the Ano5 gene

Mutations in the gene ANO5, encoding for the transmembrane protein Anoctamin 5 (Ano5), have been identified to cause gnathodiaphyseal dysplasia (GDD) in humans, a skeletal disorder characterized by sclerosis of tubular bones, increased fracture risk and fibro-osseous lesions of the jawbones. To better understand the pathomechanism of GDD we have generated via Crispr/CAS9 gene editing a mouse model harboring the murine equivalent (Ano5 p.T491F) of a GDD-causing ANO5 mutation identified in a previously reported patient. Skeletal phenotyping by contact radiography, μCT and undecalcified histomorphometry was performed in male mice, heterozygous and homozygous for the mutation, at the ages of 12 and 24 weeks. These mice did not display alterations of skeletal microarchitecture or mandible morphology. The results were confirmed in female mice and animals derived from a second, independent clone. Finally, no skeletal phenotype was observed in mice lacking ~40% of their Ano5 gene due to a frameshift mutation. Therefore, our results indicate that Ano5 is dispensable for bone homeostasis in mice, at least under unchallenged conditions, and that these animals may not present the most adequate model to study the physiological role of Anoctamin 5.ISSN:2352-187

Post-weaning epiphysiolysis causes distal femur dysplasia and foreshortened hindlimbs in fetuin-A-deficient mice

Fetuin-A / alpha(2)-Heremans-Schmid-glycoprotein (gene name Ahsg) is a systemic inhibitor of ectopic calcification. Due to its high affinity for calcium phosphate, fetuin-A is highly abundant in mineralized bone matrix. Foreshortened femora in fetuin-A-deficient Ahsg(-/-) mice indicated a role for fetuin-A in bone formation. We studied early postnatal bone development in fetuin-A-deficient mice and discovered that femora from Ahsg(-/-) mice exhibited severely displaced distal epiphyses and deformed growth plates, similar to the human disease slipped capital femoral epiphysis (SCFE). The growth plate slippage occurred in 70% of Ahsg(-/-) mice of both sexes around three weeks postnatal. At this time point, mice weaned and rapidly gained weight and mobility. Epiphysis slippage never occurred in wildtype and heterozygous Ahsg(+/-) mice. Homozygous fetuin-A-deficient Ahsg(-/-) mice and, to a lesser degree, heterozygous Ahsg(+/-) mice showed lesions separating the proliferative zone from the hypertrophic zone of the growth plate. The hypertrophic growth plate cartilage in long bones from Ahsg(-/-) mice was significantly elongated and V-shaped until three weeks of age and thus prior to the slippage. Genome-wide transcriptome analysis of laser-dissected distal femoral growth plates from 13-day-old Ahsg(-/-) mice revealed a JAK-STAT-mediated inflammatory response including a 550-fold induction of the chemokine Cxcl9. At this stage, vascularization of the elongated growth plates was impaired, which was visualized by immunofluorescence staining. Thus, fetuin-A-deficient mice may serve as a rodent model of growth plate pathologies including SCFE and inflammatory cartilage degradation

The chaperone activity of 4PBA ameliorates the skeletal phenotype of Chihuahua, a zebrafish model for dominant osteogenesis imperfecta

Classical osteogenesis imperfecta (OI) is a bone disease caused by type I collagen mutations and characterized by bone fragility, frequent fractures in absence of trauma and growth deficiency. No definitive cure is available for OI and to develop novel drug therapies, taking advantage of a repositioning strategy, the small teleost zebrafish (Danio rerio) is a particularly appealing model. Its small size, high proliferative rate, embryo transparency and small amount of drug required make zebrafish the model of choice for drug screening studies, when a valid disease model is available. We performed a deep characterization of the zebrafish mutant Chihuahua, that carries a G574D (p.G736D) substitution in the alpha 1 chain of type I collagen. We successfully validated it as a model for classical OI. Growth of mutants was delayed compared with WT. X-ray, mCT, alizarin red/alcian blue and calcein staining revealed severe skeletal deformity, presence of fractures and delayed mineralization. Type I collagen extracted from different tissues showed abnormal electrophoretic migration and low melting temperature. The presence of endoplasmic reticulum (ER) enlargement due to mutant collagen retention in osteoblasts and fibroblasts of mutant fish was shown by electron and confocal microscopy. Two chemical chaperones, 4PBA and TUDCA, were used to ameliorate the cellular stress and indeed 4PBA ameliorated bone mineralization in larvae and skeletal deformities in adult, mainly acting on reducing ER cisternae size and favoring collagen secretion. In conclusion, our data demonstrated that ER stress is a novel target to ameliorate OI phenotype; chemical chaperones such as 4PBA may be, alone or in combination, a new class of molecules to be further investigated for OI treatment

Genome-wide gene expression analysis reveals the induction of pro-inflammatory genes in growth plates of 13-day-old <i>Ahsg</i>^<i>-/-</i> mice.

<p>(A) Heatmap representation of the normalized gene expression in individual growth plates shows consistently strong induction of large gene clusters in fetuin-A deficient <i>Ahsg</i>^<i>-/-</i> mice compared to wildtype <i>Ahsg</i>^<i>+/+</i> mice. Gene expression in growth plates of <i>Ahsg</i>^<i>+/-</i> was heterogeneous and had partial overlap with both <i>Ahsg</i>^<i>-/-</i> and <i>Ahsg</i>^<i>+/+</i> genotypes. (B) Volcano plot comparing the normalized growth plate gene expression between <i>Ahsg</i>^<i>-/-</i> and <i>Ahsg</i>^<i>+/+</i> mice. The plot shows that more significantly differentially expressed genes were induced (red), and few genes were significantly repressed (blue). Genes marked with red circles were used for validation of microarray data with qRT-PCR. (C) The seven most highly differentially induced genes were validated using qRT-PCR. The graph represents the log₂ fold changes in expression in <i>Ahsg</i>^<i>-/-</i> compared to <i>Ahsg</i>^<i>+/+</i> samples. Data was analyzed using Student’s t-test: **p<0.005, ***p<0.001.</p

Growth plate histology and immunofluorescent localization of fetuin-A in distal femoral growth plates.

<p>(A) Decalcified femur paraffin sections from mice of different ages were stained with an anti-fetuin-A antibody (red) and nuclei were counterstained with DAPI (blue). In the growth plate, fetuin-A was localized in hypertrophic chondrocytes. <i>Ahsg</i>^<i>-/-</i> mice served as negative control (lower panel). (B) Similar to wildtype mice, fetuin-A was detected in hypertrophic chondrocytes from eight-week-old <i>Ahsg</i>^<i>+/-</i> mice. Fetuin-A staining was negative in growth plates containing a lesion (green arrows). (C) A magnified view of the marked area in (A) shows the cytoplasmic localization of fetuin-A in hypertrophic chondrocytes. Scale bars are 75 μm.</p

Bone dysplasia in fetuin-A deficient mice.

<p>(A) Growth curves of femora from both, male and female <i>Ahsg</i>^<i>+/+</i>, <i>Ahsg</i>^<i>+/-</i> and <i>Ahsg</i>^<i>-/-</i> mice reveal that femora of <i>Ahsg</i>^<i>-/-</i> mice were significantly shortened compared to their littermates at the age of four weeks and older. Error bars show SD. Data was analyzed by One-Way ANOVA: *p<0.05, **p<0.01, ***p<0.001. (B) Measurement of distal femur width shows a widening of the distal femoral epiphysis in <i>Ahsg</i>^<i>-/-</i> mice at the age of four weeks and older. (C) Three-dimensional reconstructions from μCT measurements of eight-week-old male mice and (D) matching 2D μCT cross-sections. Micro-CT analysis shows a posterior rotation of the distal femoral epiphysis in <i>Ahsg</i>^<i>-/-</i> mice. (E) The angle of the distal femoral epiphysis relative to the shaft (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187030#pone.0187030.s001" target="_blank">S1 Fig</a>) was significantly decreased in male <i>Ahsg</i>^<i>-/-</i> mice. Error bars show SD. Data was analyzed by Student’s t-test: ***p<0.001. (F) Prevalence of the dysplasia in male and female C57BL/6 <i>Ahsg</i>^<i>-/-</i> mice. Mice at the age of four weeks and older were evaluated for dysplasia by either bone length measurements from isolated bones or from radiographic images. Out of 62 <i>Ahsg</i>^<i>-/-</i> mice, 44 showed dysplasia in their distal femur in one or both legs, 18 mice had no obvious phenotype anomaly.</p