BackgroundThe identification of strategies to improve mutant CFTR function remains a key priority in the development of new treatments for cystic fibrosis (CF). Previous studies demonstrated that the K+ channel opener 1-ethyl-2-benzimidazolone (1-EBIO) potentiates CFTR-mediated Cl− secretion in cultured cells and mouse colon. However, the effects of 1-EBIO on wild-type and mutant CFTR function in native human colonic tissues remain unknown.MethodsWe studied the effects of 1-EBIO on CFTR-mediated Cl− secretion in rectal biopsies from 47 CF patients carrying a wide spectrum of CFTR mutations and 57 age-matched controls. Rectal tissues were mounted in perfused micro-Ussing chambers and the effects of 1-EBIO were compared in control tissues, CF tissues expressing residual CFTR function and CF tissues with no detectable Cl− secretion.ResultsStudies in control tissues demonstrate that 1-EBIO activated CFTR-mediated Cl− secretion in the absence of cAMP-mediated stimulation and potentiated cAMP-induced Cl− secretion by 39.2±6.7% (P<0.001) via activation of basolateral Ca2+-activated and clotrimazole-sensitive KCNN4 K+ channels. In CF specimens, 1-EBIO potentiated cAMP-induced Cl− secretion in tissues with residual CFTR function by 44.4±11.5% (P<0.001), but had no effect on tissues lacking CFTR-mediated Cl−conductance.ConclusionsWe conclude that 1-EBIO potentiates Cl−secretion in native CF tissues expressing CFTR mutants with residual Cl− channel function by activation of basolateral KCNN4 K+ channels that increase the driving force for luminal Cl− exit. This mechanism may augment effects of CFTR correctors and potentiators that increase the number and/or activity of mutant CFTR channels at the cell surface and suggests KCNN4 as a therapeutic target for CF.
BackgroundOsteosarcoma (OS) is the most common pediatric primary malignant bone tumor. As the prognosis for patients following standard treatment did not improve for almost three decades, functional preclinical models that closely reflect important clinical cancer characteristics are urgently needed to develop and evaluate new treatment strategies. The objective of this study was to establish an orthotopic xenotransplanted mouse model using patient-derived tumor tissue.MethodsFresh tumor tissue from an adolescent female patient with osteosarcoma after relapse was surgically xenografted into the right tibia of 6 immunodeficient BALB/c Nu/Nu mice as well as cultured into medium. Tumor growth was serially assessed by palpation and with magnetic resonance imaging (MRI). In parallel, a primary cell line of the same tumor was established. Histology and high-resolution array-based comparative genomic hybridization (aCGH) were used to investigate both phenotypic and genotypic characteristics of different passages of human xenografts and the cell line compared to the tissue of origin.ResultsA primary OS cell line and a primary patient-derived orthotopic xenotranplanted mouse model were established. MRI analyses and histopathology demonstrated an identical architecture in the primary tumor and in the xenografts. Array-CGH analyses of the cell line and all xenografts showed highly comparable patterns of genomic progression. So far, three further primary patient-derived orthotopic xenotranplanted mouse models could be established.ConclusionWe report the first orthotopic OS mouse model generated by transplantation of tumor fragments directly harvested from the patient. This model represents the morphologic and genomic identity of the primary tumor and provides a preclinical platform to evaluate new treatment strategies in OS.
BackgroundHistone 3.3 (H3.3) hotspot mutations in bone tumors occur in the vast majority of giant cell tumors of bone (GCTBs; 96%), chondroblastomas (95%) and in a few cases of osteosarcomas. However, clinical presentation, histopathological features, and additional molecular characteristics of H3.3 mutant osteosarcomas are largely unknown.MethodsIn this multicentre, retrospective study, a total of 106 conventional high-grade osteosarcomas, across all age groups were re-examined for hotspot mutations in the H3.3 coding genes H3F3A and H3F3B. H3.3 mutant osteosarcomas were re-evaluated in a multidisciplinary manner and analyzed for genome-wide DNA-methylation patterns and DNA copy number aberrations alongside H3.3 wild-type osteosarcomas and H3F3A G34W/L mutant GCTBs.ResultsSix osteosarcomas (6/106) carried H3F3A hotspot mutations. No mutations were found in H3F3B. All patients with H3F3A mutant osteosarcoma were older than 30 years with a median age of 65 years. Copy number aberrations that are commonly encountered in high-grade osteosarcomas also occurred in H3F3A mutant osteosarcomas. Unlike a single osteosarcoma with a H3F3A K27M mutation, the DNA methylation profiles of H3F3A G34W/R mutant osteosarcomas were clearly different from H3.3 wild-type osteosarcomas, but more closely related to GCTBs. The most differentially methylated promoters between H3F3A G34W/R mutant and H3.3 wild-type osteosarcomas were in KLLN/PTEN (p < 0.00005) and HIST1H2BB (p < 0.0005).ConclusionsH3.3 mutations in osteosarcomas may occur in H3F3A at mutational hotspots. They are overall rare, but become more frequent in osteosarcoma patients older than 30 years. Osteosarcomas carrying H3F3A G34W/R mutations are associated with epigenetic dysregulation of KLLN/PTEN and HIST1H2BB.Electronic supplementary materialThe online version of this article (doi:10.1186/s13569-017-0075-5) contains supplementary material, which is available to authorized users.
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