Rebecca Evans

Three-Dimensional Microfluidic Tri-Culture Model of the Bone Marrow Microenvironment for Study of Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) initiates and progresses in the bone marrow, and as such, the marrow microenvironment is a critical regulatory component in development of this cancer. However, ALL studies were conducted mainly on flat plastic substrates, which do not recapitulate the characteristics of marrow microenvironments. To study ALL in a model of in vivo relevance, we have engineered a 3-D microfluidic cell culture platform. Biologically relevant populations of primary human bone marrow stromal cells, osteoblasts and human leukemic cells representative of an aggressive phenotype were encapsulated in 3-D collagen matrix as the minimal constituents and cultured in a microfluidic platform. The matrix stiffness and fluidic shear stress were controlled in a physiological range. The 3-D microfluidic as well as 3-D static models demonstrated coordinated cell-cell interactions between these cell types compared to the compaction of the 2-D static model. Tumor cell viability in response to an antimetabolite chemotherapeutic agent, cytarabine in tumor cells alone and tri-culture models for 2-D static, 3-D static and 3-D microfluidic models were compared. The present study showed decreased chemotherapeutic drug sensitivity of leukemic cells in 3-D tri-culture models from the 2-D models. The results indicate that the bone marrow microenvironment plays a protective role in tumor cell survival during drug treatment. The engineered 3-D microfluidic tri-culture model enables systematic investigation of effects of cell-cell and cell-matrix interactions on cancer progression and therapeutic intervention in a controllable manner, thus improving our limited comprehension of the role of microenvironmental signals in cancer biology.

UK Renal Registry 18th Annual Report 2015

The UK Renal Registry (UKRR) continues to provide a national source of NHS healthcare data on patients dependent on renal replacement therapy (RRT) across the four nations. Using electronic reporting and substantial integration across the 71 adult and 13 paediatric renal centres independent audit and analysis of dialysis and transplant activity and care across the UK is provided. The UKRR is part of the UK Renal Association and is funded directly by participating renal centres through an annual capitation fee per patient per annum, currently £19 or 0.01% of annual RRT running costs. The UKRR remains relatively unique amongst renal registries in publishing both centre-specific analyses of indicators of quality of care, such as haemoglobin and also ageadjusted survival statistics for each renal centre [1].

Bone Marrow Microenvironment Niche Regulates miR-221/222 in Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) has many features in common with normal B-cell progenitors, including their ability to respond to diverse signals from the bone marrow microenvironment (BMM) resulting in regulation of cell-cycle progression and survival. Bone marrow–derived cues influence many elements of both steady state hematopoiesis and hematopoietic tumor cell phenotypes through modulation of gene expression. miRNAs are one regulatory class of small noncoding RNAs that have been shown to be increasingly important in diverse settings of malignancy. In the current study, miRNA profiles were globally altered in ALL cells following exposure to primary human bone marrow niche cells, including bone marrow stromal cells (BMSC) and primary human osteoblasts (HOB). Specifically, mature miR-221 and miR-222 transcripts were decreased in ALL cells cocultured with BMSC or HOB, coincident with increased p27 (CDKN1B), a previously validated target. Increased p27 protein in ALL cells exposed to BMSC or HOB is consistent with accumulation of tumor cells in the G0 phase of the cell cycle and resistance to chemotherapy-induced death. Overexpression of miR-221 in ALL cells during BMSC or HOB coculture prompted cell-cycle progression and sensitization of ALL cells to cytotoxic agents, blunting the protective influence of the BMM. These novel observations indicate that BMM regulation of miR-221/222 contributes to marrow niche-supported tumor cell quiescence and survival of residual cells. Implications: Niche-influenced miR-221/222 may define a novel therapeutic target in ALL to be combined with existing cytotoxic agents to more effectively eradicate refractory disease that contributes to relapse. Mol Cancer Res; 14(10); 909–19. ©2016 AACR.

In Vitro Expansion of Bone Marrow Derived Mesenchymal Stem Cells Alters DNA Double Strand Break Repair of Etoposide Induced DNA Damage

Mesenchymal stem cells (MSCs) are of interest for use in diverse cellular therapies. Ex vivo expansion of MSCs intended for transplantation must result in generation of cells that maintain fidelity of critical functions. Previous investigations have identified genetic and phenotypic alterations of MSCs with in vitro passage, but little is known regarding how culturing influences the ability of MSCs to repair double strand DNA breaks (DSBs), the most severe of DNA lesions. To investigate the response to DSB stress with passage in vitro, primary human MSCs were exposed to etoposide (VP16) at various passages with subsequent evaluation of cellular damage responses and DNA repair. Passage number did not affect susceptibility to VP16 or the incidence and repair kinetics of DSBs. Nonhomologous end joining (NHEJ) transcripts showed little alteration with VP16 exposure or passage; however, homologous recombination (HR) transcripts were reduced following VP16 exposure with this decrease amplified as MSCs were passaged in vitro. Functional evaluations of NHEJ and HR showed that MSCs were unable to activate NHEJ repair following VP16 stress in cells after successive passage. These results indicate that ex vivo expansion of MSCs alters their ability to perform DSB repair, a necessary function for cells intended for transplantation.

Quality of Reporting and Study Design of CKD Cohort Studies Assessing Mortality in the Elderly Before and After STROBE: A Systematic Review

Background The STrengthening the Reporting of OBservational studies in Epidemiology (STROBE) statement was published in October 2007 to improve quality of reporting of observational studies. The aim of this review was to assess the impact of the STROBE statement on observational study reporting and study design quality in the nephrology literature. Study Design Systematic literature review. Setting & Population European and North American, Pre-dialysis Chronic Kidney Disease (CKD) cohort studies. Selection Criteria for Studies Studies assessing the association between CKD and mortality in the elderly (>65 years) published from 1st January 2002 to 31st December 2013 were included, following systematic searching of MEDLINE & EMBASE. Predictor Time period before and after the publication of the STROBE statement. Outcome Quality of study reporting using the STROBE statement and quality of study design using the Newcastle Ottawa Scale (NOS), Scottish Intercollegiate Guidelines Network (SIGN) and Critical Appraisal Skills Programme (CASP) tools. Results 37 papers (11 Pre & 26 Post STROBE) were identified from 3621 potential articles. Only four of the 22 STROBE items and their sub-criteria (objectives reporting, choice of quantitative groups and description of and carrying out sensitivity analysis) showed improvements, with the majority of items showing little change between the period before and after publication of the STROBE statement. Pre- and post-period analysis revealed a Manuscript STROBE score increase (median score 77.8% (Inter-quartile range [IQR], 64.7–82.0) vs 83% (IQR, 78.4–84.9, p = 0.05). There was no change in quality of study design with identical median scores in the two periods for NOS (Manuscript NOS score 88.9), SIGN (Manuscript SIGN score 83.3) and CASP (Manuscript CASP score 91.7) tools. Limitations Only 37 Studies from Europe and North America were included from one medical specialty. Assessment of study design largely reliant on good reporting. Conclusions This study highlights continuing deficiencies in the reporting of STROBE items and their sub-criteria in cohort studies in nephrology. There was weak evidence of improvement in the overall reporting quality, with no improvement in methodological quality of CKD cohort studies between the period before and after publication of the STROBE statement.

BCL6 modulation of acute lymphoblastic leukemia response to chemotherapy

The bone marrow niche has a significant impact on acute lymphoblastic leukemia (ALL) cell phenotype. Of clinical relevance is the frequency with which quiescent leukemic cells, in this niche, survive treatment and contribute to relapse. This study suggests that marrow microenvironment regulation of BCL6 in ALL is one factor that may be involved in the transition between proliferative and quiescent states of ALL cells. Utilizing ALL cell lines, and primary patient tumor cells we observed that tumor cell BCL6 protein abundance is decreased in the presence of primary human bone marrow stromal cells (BMSC) and osteoblasts (HOB). Chemical inhibition, or shRNA knockdown, of BCL6 in ALL cells resulted in diminished ALL proliferation. As many chemotherapy regimens require tumor cell proliferation for optimal efficacy, we investigated the consequences of constitutive BCL6 expression in leukemic cells during co-culture with BMSC or HOB. Forced chronic expression of BCL6 during co-culture with BMSC or HOB sensitized the tumor to chemotherapy induced cell death. Combination treatment of caffeine, which increases BCL6 expression in ALL cells, with chemotherapy extended the event free survival of mice. These data suggest that BCL6 is one factor, modulated by microenvironment derived cues that may contribute to regulation of ALL therapeutic response.

Modeling The Bone Marrow Microenvironment’s Influence on Leukemic Disease

The bone marrow microenvironment serves as both the site of initiation of the majority of hematopoietic malignancies and also contributes to maintenance of minimal residual disease by promoting biologically relevant changes in tumor cells. These functional alterations of leukemic cells include, but are not limited to modulation of cell cycle, regulation of anti-apoptotic signaling cascades, and influence on metabolic activity. Of clinical relevance, these pathways impact on therapeutic response, making it critical to have robust in vitro systems to interrogate tumor cell interactions with stromal elements of the microenvironment to screen chemotherapeutic agents and inform the in vivo model design.