Metronomic doses and drug schematic combination response tested within chambered coverslips for the treatment of breast cancer cells (JIMT-1)

Low-dose metronomic (LDM) chemotherapy is an alternative to conventional chemotherapy and is the most frequently used approach in low dose chemotherapy regimens. The selection of patients, drug dosages, and dosing intervals in LDM is empirical. In this study, we systematically examined the schedule-dependent interaction of drugs on a breast cancer cell line (BCC) cultured in chambered coverslips. The LDM studies were combined with cell staining in order to better characterize different cell states and cell death modes, including caspase-dependent apoptosis, caspase-independent cell death and autophagy-dependent cell death. Microscope images were examined using the Fiji Trainable Weka Segmentation plugin to analyse cell area in 7500 images showing different modes of cell death. Paclitaxel combined with LDM chemotherapy demonstrated a reduction in the area covered by live cells. In contrast, there was an induction of high levels of cell death due to caspase-dependent apoptosis.Fil: Rosero, Gustavo. Universidad Tecnológica Nacional; Argentina. Albert Ludwigs University of Freiburg; AlemaniaFil: Pattarone, Gisela. Albert Ludwigs University of Freiburg; AlemaniaFil: Peñaherrera Pazmiño, Ana Belén. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Tecnológica Nacional; Argentina. Albert Ludwigs University of Freiburg; AlemaniaFil: Pilz, Julia. Albert Ludwigs University of Freiburg; AlemaniaFil: Bödecker, Joschka. Albert Ludwigs University of Freiburg; AlemaniaFil: Perez, Maximiliano Sebastian. Universidad de Buenos Aires; Argentina. Universidad Tecnológica Nacional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mertelsmann, Roland. Albert Ludwigs University of Freiburg; AlemaniaFil: Lerner, Betiana. Universidad de Buenos Aires; Argentina. Universidad Tecnológica Nacional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Follo, Marie. Universidad de Buenos Aires; Argentin

Microscopic evaluation of tongue dorsum biofilm from halitosis patients: an ex vivo study using confocal laser scanning microscopy (CLSM)

A category of oral biofilm which is still not well understood is the one coating the tongue, although various reports have associated its presence with halitosis in patients (1). The aim of the study was to visualize the three-dimensional bacteria distribution within the biofilm in order to better understand the ecological balance which regulates it. Tongue plaque samples from four halitosis-diagnosed patients and four healthy volunteers were analysed and compared. The biofilm was collected using a 0.1ml sterile inoculating loop. The visualization of the tongue dorsum biofilm was performed combining fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) (2). Eubacteria, Streptococcus spp. and Fusobacterium nucleatum were stained using specific fluorescent genetic probes. Morphological analysis by CLSM illustrated the different distribution of the species which were tracked: Streptococcus spp. appeared immerged within the samples, while F. nucleatum was found in the peripheral areas of the samples. Furthermore, F. nucleatum appeared to exist without the presence of the Streptococcus spp. in the halitosis group. This study showed the architecture of tongue dorsum biofilm by means of imaging techniques, highlighting the distribution of the tracked bacterial species within the biofilm sample of the plaque.The authors are grateful to Dr. A. Zurcher and to Mr. G. Heuzeroth, University of Basel, for their help in the recruiting and sampling procedures

Negative correlation of single-cell PAX3:FOXO1 expression with tumorigenicity in rhabdomyosarcoma

Rhabdomyosarcomas (RMS) are phenotypically and functionally heterogeneous. Both primary human RMS cultures and low-passage Myf6Cre,Pax3:Foxo1,p53 mouse RMS cell lines, which express the fusion oncoprotein Pax3:Foxo1 and lack the tumor suppressor Tp53 (Myf6Cre,Pax3:Foxo1,p53), exhibit marked heterogeneity in PAX3:FOXO1 (P3F) expression at the single cell level. In mouse RMS cells, P3F expression is directed by the Pax3 promoter and coupled to eYFP. YFPlow/P3Flow mouse RMS cells included 87% G0/G1 cells and reorganized their actin cytoskeleton to produce a cellular phenotype characterized by more efficient adhesion and migration. This translated into higher tumor-propagating cell frequencies of YFPlow/P3Flow compared with YFPhigh/P3Fhigh cells. Both YFPlow/P3Flow and YFPhigh/P3Fhigh cells gave rise to mixed clones in vitro, consistent with fluctuations in P3F expression over time. Exposure to the anti-tropomyosin compound TR100 disrupted the cytoskeleton and reversed enhanced migration and adhesion of YFPlow/P3Flow RMS cells. Heterogeneous expression of PAX3:FOXO1 at the single cell level may provide a critical advantage during tumor progression

Cost-effective fabrication of photopolymer molds with multi-level microstructures for pdms microfluidic device manufacture

This paper describes a methodology of photopolymer mold fabrication with multi-level microstructures for polydimethylsiloxane (PDMS) microfluidic device manufacture. Multi-level microstructures can be performed by varying UVA exposure time and channel width. Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM) and profilometry techniques have been employed to characterize the molds. Multiple molds with multi-level microstructures can be formed in a unique piece. Overall height/depth of the structures reaches up to 677 μm and a minimum of 21 μm. The method provides several advantages such as reduction of fabrication time, multiple structures with diverse topologies, a great variety of depth and height in a single mold and low cost of fabrication. The effectiveness of multi-level microstructure fabrication was evaluated by constructing PDMS microfluidic devices for cell culture and proliferation.Fil: Olmos Carreno, Carol Maritza. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Penãherrera, Ana. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Albert Ludwigs University of Freiburg; AlemaniaFil: Rosero Yánez, Gustavo Ivan. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Albert Ludwigs University of Freiburg; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vizuete, Karla. Universidad de Las Fuerzas Armadas; EcuadorFil: Ruarte, Darío. Albert Ludwigs University of Freiburg; AlemaniaFil: Follo, Marie. Albert Ludwigs University of Freiburg; AlemaniaFil: Vaca Mora, Andrea Vanessa. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Arroyo, Carlos R.. Universidad de Las Fuerzas Armadas; EcuadorFil: Debut, Alexis. Universidad de Las Fuerzas Armadas; EcuadorFil: Cumbal Flores, Luis. Universidad de Las Fuerzas Armadas; EcuadorFil: Perez, Maximiliano Sebastian. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Florida International University; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lerner, Betiana. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Florida International University; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mertelsmann, Roland. Albert Ludwigs University of Freiburg; Alemani

Biodegradable Nanocarriers Resembling Extracellular Vesicles Deliver Genetic Material with the Highest Efficiency to Various Cell Types

Efficient delivery of genetic material to primary cells remains challenging. Here, efficient transfer of genetic material is presented using synthetic biodegradable nanocarriers, resembling extracellular vesicles in their biomechanical properties. This is based on two main technological achievements: generation of soft biodegradable polyelectrolyte capsules in nanosize and efficient application of the nanocapsules for co‐transfer of different RNAs to tumor cell lines and primary cells, including hematopoietic progenitor cells and primary T cells. Near to 100% efficiency is reached using only 2.5 × 10−4 pmol of siRNA, and 1 × 10−3 nmol of mRNA per cell, which is several magnitude orders below the amounts reported for any of methods published so far. The data show that biodegradable nanocapsules represent a universal and highly efficient biomimetic platform for the transfer of genetic material with the utmost potential to revolutionize gene transfer technology in vitro and in vivo

ROCK1/2 signaling contributes to corticosteroid-refractory acute graft-versus-host disease

Patients with corticosteroid-refractory acute graft-versus-host disease (aGVHD) have a low one-year survival rate. Identification and validation of novel targetable kinases in patients who experience corticosteroid-refractory-aGVHD may help improve outcomes. Kinase-specific proteomics of leukocytes from patients with corticosteroid-refractory-GVHD identified rho kinase type 1 (ROCK1) as the most significantly upregulated kinase. ROCK1/2 inhibition improved survival and histological GVHD severity in mice and was synergistic with JAK1/2 inhibition, without compromising graft-versus-leukemia-effects. ROCK1/2-inhibition in macrophages or dendritic cells prior to transfer reduced GVHD severity. Mechanistically, ROCK1/2 inhibition or ROCK1 knockdown interfered with CD80, CD86, MHC-II expression and IL-6, IL-1β, iNOS and TNF production in myeloid cells. This was accompanied by impaired T cell activation by dendritic cells and inhibition of cytoskeletal rearrangements, thereby reducing macrophage and DC migration. NF-κB signaling was reduced in myeloid cells following ROCK1/2 inhibition. In conclusion, ROCK1/2 inhibition interferes with immune activation at multiple levels and reduces acute GVHD while maintaining GVL-effects, including in corticosteroid-refractory settings

Phosphorylation of BECLIN-1 by BCR-ABL suppresses autophagy in chronic myeloid leukemia

Autophagy is a genetically regulated process of adaptation to metabolic stress and was recently shown to be involved in the treatment response of chronic myeloid leukemia (CML). However, in vivo data are limited and the molecular mechanism of autophagy regulators in the process of leukemogenesis is not completely understood. Here we show that Beclin-1 knockdown, but not Atg5 deletion in a murine CML model leads to a reduced leukemic burden and results in a significantly prolonged median survival of targeted mice. Further analyses of murine cell lines and primary patient material indicate that active BCR-ABL directly interacts with BECLIN-1 and phosphorylates its tyrosine residues 233 and 352, resulting in autophagy suppression. By using phosphorylation-deficient and phosphorylation-mimic mutants, we identify BCR-ABL induced BECLIN-1 phosphorylation as a crucial mechanism for BECLIN-1 complex formation: interaction analyses exhibit diminished binding of the positive autophagy regulators UVRAG, VPS15, ATG14 and VPS34 and enhanced binding of the negative regulator Rubicon to BCR-ABL-phosphorylated BECLIN-1. Taken together, our findings show interaction of BCR-ABL and BECLIN-1 thereby highlighting the importance of BECLIN-1-mediated autophagy in BCR-ABL+ cells

The Nlrp3 inflammasome regulates acute graft-versus-host disease

The success of allogeneic hematopoietic cell transplantation is limited by acute graft-versus-host disease (GvHD), a severe complication accompanied by high mortality rates. Yet, the molecular mechanisms initiating this disease remain poorly defined. In this study, we show that, after conditioning therapy, intestinal commensal bacteria and the damage-associated molecular pattern uric acid contribute to Nlrp3 inflammasome-mediated IL-1β production and that gastrointestinal decontamination and uric acid depletion reduced GvHD severity. Early blockade of IL-1β or genetic deficiency of the IL-1 receptor in dendritic cells (DCs) and T cells improved survival. The Nlrp3 inflammasome components Nlrp3 and Asc, which are required for pro-IL-1β cleavage, were critical for the full manifestation of GvHD. In transplanted mice, IL-1β originated from multiple intestinal cell compartments and exerted its effects on DCs and T cells, the latter being preferentially skewed toward Th17. Compatible with these mouse data, increased levels of active caspase-1 and IL-1β were found in circulating leukocytes and intestinal GvHD lesions of patients. Thus, the identification of a crucial role for the Nlrp3 inflammasome sheds new light on the pathogenesis of GvHD and opens a potential new avenue for the targeted therapy of this severe complication

Existence of reprogrammed lymphoma stem cells in a murine ALCL-like model.

Funder: DJCLS (R14/22) MSCA-ITN-2015-ETN AlkatrasFunder: DFG (CRC850)Funder: DFG (CRC850) BMBF (DeCaRe, FKZ 01ZX1409B)Funder: DJCLS (R14/22) MSCA-ITN-2015-ETN Alkatras DFG (FOR 2033 B1)Funder: DJCLS (R14/22) MSCA-ITN-2015-ETN Alkatras Grant from the Government of Baden-Württemberg (BSL)While cancer stem cells are well established in certain hematologic and solid malignancies, their existence in T cell lymphoma is unclear and the origin of disease is not fully understood. To examine the existence of lymphoma stem cells, we utilized a mouse model of anaplastic large cell lymphoma. Established NPM-ALK+ lymphomas contained heterogeneous cell populations ranging from mature T cells to undifferentiated hematopoietic stem cells. Interestingly, CD4-/CD8- double negative (DN) lymphoma cells aberrantly expressed the T cell receptor α/β chain. Serial transplantation of sorted CD4/CD8 and DN lymphoma subpopulations identified lymphoma stem cells within the DN3/DN4 T cell population, whereas all other subpopulations failed to establish serial lymphomas. Moreover, transplanted lymphoma DN3/DN4 T cells were able to differentiate and gave rise to mature lymphoma T cells. Gene expression analyses unmasked stem-cell-like transcriptional regulation of the identified lymphoma stem cell population. Furthermore, these lymphoma stem cells are characterized by low CD30 expression levels, which might contribute to limited long-term therapeutic success in patients treated with anti-CD30-targeted therapies. In summary, our results highlight the existence of a lymphoma stem cell population in a NPM-ALK-driven CD30+ mouse model, thereby giving the opportunity to test innovative treatment strategies developed to eradicate the origin of disease