Biology subjects

Barker, K.

Publications and source records attributed to Barker, K..

7 recordsLinked to original sources

bioRxiv2026

A COJEC-chemotherapy resistant model of Th-ALK(F1174L)/MYCN neuroblastoma offers insights into tumour immune evasion and development of the bone marrow metastatic niche

Multi-agent COJEC chemotherapy is the main-stay of induction treatment for patients diagnosed with high-risk neuroblastoma. However, at least 10% of patients will be primary refractory to chemotherapy and only 50% achieve 5-year overall survival. The bone marrow is the most frequent site of metastasis in these patients. Novel approaches are required to improve response rates but the inter- and intra- patient tumour heterogeneity and dynamics of the neuroblastoma immune microenvironment makes anticipation of resistance phenotypes incredibly challenging. We present here a novel immunocompetent C57 Bl/6 model of Th-ALK(F1174L)/MYCN neuroblastoma, in which spontaneous abdominal tumours are driven by expression of mutant Anaplastic Lymphoma Kinase and over-expression of Mycn in the neural crest. We have used this model to generate a personalised dosing schedule inducing COJEC-chemotherapy resistance, in which individual mice receive chemotherapy cycles dependent upon the progression of their neuroblastoma tumours. Using both single cell RNA sequencing and spatial immunophenotyping gave us extraordinary precision in our comprehensive analysis of the tumour intrinsic and microenvironmental factors associated with COJEC resistance. We found that the resistance phenotype was driven by Cdk8 upregulation in adrenergic and mesenchymal tumour cells. Infiltration of immunosuppressive myeloid-derived immune cells and remodeling of the tumour-associated stroma further contributed to COJEC resistance. In the bone marrow we observed expansion of neutrophils and evidence of NETosis associated with micro-metastatic disease. Our results further endorse the development of CDK8-targeting therapeutics for neuroblastoma patients which might boost the anti-tumour immune response. Additional studies will be required to define the roles of neutrophils and neutrophil NETosis in neuroblastoma progression and metastasis. Our C57 Bl/6 model will be pivotal in future preclinical studies of immune-modulating therapeutics.

cancer biology↗

bioRxiv2025

Differential chromatin response to retinoic acid in neuroblastoma according to type of ATRX mutation

Neuroblastoma is a childhood cancer, arising in the developing sympathetic nervous system. Differentiation therapy with 13-cis-retinoic acid (RA) is routinely given to children with high-risk neuroblastoma in the minimal residual disease setting to prevent relapse, however there is little understanding of which patients benefit from RA therapy. ATRX alterations are identified in 10% of high-risk neuroblastomas and associated with poor outcomes. The commonest type of ATRX alterations in neuroblastoma are in-frame multi-exon deletions, followed by nonsense mutations predicted to result in loss-of-function (ATRX LoF). We treated paired ATRX wild-type and ATRX LoF neuroblastoma cell lines with RA and show that cells with ATRX LoF fail to upregulate direct RA target genes. Cells with ATRX LoF also show reduced chromatin accessibility at genes involved in differentiation and development following RA treatment. Conversely, neuroblastoma models with in-frame deletions mount a response to RA and show in-vitro sensitivity to RA. Taken together this shows that the mechanism of differentiation in ATRX-altered neuroblastoma depends on the type of ATRX alteration, with implications relating to both oncogenesis and therapeutic response.

cancer biology↗

bioRxiv2022

COMBINATION THERAPIES TARGETING ALK-ABERRANT NEUROBLASTOMA IN PRECLINICAL MODELS.

BackgroundALK activating mutations are identified in approximately 10% of newly diagnosed neuroblastomas and ALK amplifications in a further 1-2% of cases. Lorlatinib, a third generation ALK inhibitor, will soon be given alongside induction chemotherapy for children with ALK-aberrant neuroblastoma. However, resistance to single agent treatment has been reported and therapies that improve the response duration are urgently required. We studied the preclinical combination of lorlatinib with chemotherapy, or with the MDM2 inhibitor, idasanutlin, as recent data has suggested that ALK inhibitor resistance can be overcome through activation of the p53-MDM2 pathway. AimsTo study the preclinical activity of ALK inhibitors alone and in combination with chemotherapy or idasanutlin. MethodsWe compared different ALK inhibitors in preclinical models prior to evaluating lorlatinib in combination with chemotherapy or idasanutlin. We developed a triple chemotherapy (CAV: cyclophosphamide, doxorubicin and vincristine) in vivo dosing schedule and applied this to both neuroblastoma genetically engineered mouse models (GEMM) and patient derived xenografts (PDX). ResultsLorlatinib in combination with chemotherapy was synergistic in immunocompetent neuroblastoma GEMM. Significant growth inhibition in response to lorlatinib was only observed in the ALK-amplified PDX model with the highest ALK expression. In this PDX lorlatinib combined with idasanutlin resulted in complete tumor regression and significantly delayed tumor regrowth. ConclusionOur study suggests that in neuroblastoma, high ALK expression could be associated with response to lorlatinib and either chemotherapy or idasanutlin. The synergy between MDM2 inhibition and ALK inhibition warrants further evaluation of this combination as a potential clinical approach for children with neuroblastoma. STATEMENT OF TRANSLATIONAL RELEVANCENeuroblastoma is a pediatric tumor of the developing sympathetic nervous system. Around 50% of high-risk neuroblastoma patients are curable. Mutations or amplification of Anaplastic Lymphoma Kinase (ALK) have emerged as a marker with which to further risk-stratify patients. The ALK inhibitor lorlatinib will soon be used alongside chemotherapy in upfront treatment of high-risk patients with ALK-aberrant disease. In this preclinical study, we used a panel of ALK aberrant neuroblastoma models to evaluate ALK inhibitors focusing on lorlatinib in combination with conventional chemotherapy and the small molecule MDM2 inhibitor idasanutlin. In both approaches we found synergy in models with high basal ALK expression without MAPK pathway alterations. We conclude that in neuroblastoma the level of ALK expression could be an additional biomarker predictive of clinical response to ALK inhibitors.

cancer biology↗

bioRxiv2022

A method for chronic and semi-chronic microelectrode array implantation in deep brain structures using image guided neuronavigation

Precise targeting of deep brain structures in humans and large animal models has been a challenge for neuroscientists. Conventional protocols used in animal models typically require large access chambers which are prone to infection and involve assembly and implantation of complex microdrives for semi-chronic applications. Here we present a methodology for improving targeting of subcortical structures in large animals such as macaque monkeys, using image guided neuronavigation. Design of custom cranial caps allowed for incorporation of stable fiducial markers, required for increased targeting accuracy in neuronavigation procedures, resulting in an average targeting error of 1.6 mm over three implantations. Incorporation of anchor bolt chambers, commonly used in human neurosurgery, provided a minimally invasive entrance to the brain parenchyma, allowing for chronic recordings. By leveraging existing 3D printing technology, we fabricated an anchor bolt-mounted microdrive for semi-chronic applications. Our protocol leverages commercially available tools for implantation, decreases the risk of infection and complications of open craniotomies, and improves the accuracy and precision of chronic electrode implantations targeting deep brain structures in large animal models.

neuroscience↗

bioRxiv2020

MicroRNAs Restrain Proliferation in BRAFV600E Melanocytic Nevi

Benign melanocytic nevi frequently emerge when an acquired BRAFV600E mutation triggers unchecked proliferation and subsequent arrest in melanocytes. Recent observations have challenged the role of oncogene-induced senescence in melanocytic nevus formation, necessitating investigations into alternative mechanisms for the establishment and maintenance of proliferation arrest in nevi. We compared the transcriptomes of melanocytes from healthy human skin, nevi, and melanomas arisen from nevi and identified a set of microRNAs as highly expressed nevus-enriched transcripts. Two of these microRNAs - MIR211-5p and MIR328-3p - induced mitotic failure, genome duplication and proliferation arrest in human melanocytes through convergent targeting of AURKB. We demonstrate that BRAFV600E induces a similar proliferation arrest in primary human melanocytes that is both reversible and conditional. Specifically, BRAFV600E expression stimulates either arrest or proliferation depending on the differentiation state of the melanocyte. We report genome duplication in human melanocytic nevi, reciprocal expression of AURKB and microRNAs in nevi and melanomas, and rescue of arrested human nevus cells with AURKB expression. Together, our data describe an alternative molecular mechanism for melanocytic nevus formation that is congruent with both experimental and clinical observations.

cancer biology↗

bioRxiv2019

High multiplex, digital spatial profiling of proteins and RNA in fixed tissue using genomic detection methods

We have developed Digital Spatial Profiling (DSP), a non-destructive method for high-plex spatial profiling of proteins and RNA, using oligonucleotide detection technologies with unlimited multiplexing capability. The key breakthroughs underlying DSP are threefold: (1) multiplexed readout of proteins/RNA using oligo-tags; (2) oligo-tags attached to affinity reagents (antibodies/RNA probes) through a photocleavable (PC) linker; (3) photocleaving light projected onto the tissue sample to release PC-oligos in any spatial pattern. Here we show precise analyte reproducibility, validation, and cellular resolution using DSP. We also demonstrate biological proof-of-concept using lymphoid, colorectal tumor, and autoimmune tissue as models to profile immune cell populations, stroma, and cancer cells to identify factors specific for the diseased microenvironment. DSP utilizes the unlimited multiplexing capability of modern genomic approaches, while simultaneously providing spatial context of protein and RNA to examine biological questions based on analyte location and distribution.

pathology↗

bioRxiv2017

Methods for chair restraint and training of the common marmoset on oculomotor tasks

The oculomotor system is the most thoroughly understood sensorimotor system in the brain, due in large part to electrophysiological studies carried out in macaque monkeys trained to perform ocuolomotor tasks. A disadvantage of the macaque model is that many cortical oculomotor areas of interest lie within sulci, making high-density array and laminar recordings impractical. Further, many techniques of molecular biology developed in rodents, such as transgenic animals and optogenetic manipulation of neuronal subtypes, are limited in this species. The common marmoset (Callithrix jacchus) may potentially bridge the gap between systems neuroscience in macaques and molecular techniques, and additionally possesses a smooth cortex allowing easy access to frontoparietal oculomotor areas. To date, techniques for restraint and training of these animals to perform oculomotor tasks remain in an early stage of development. Here we provide details of a custom-designed chair for restraint of marmosets, a combination head restraint/recording chamber providing stability suitable for eye movement and neural recordings, and a training protocol for oculomotor tasks. As proof-of-principle, we report the results of a psychophysical study in marmosets trained to perform a saccade task using these methods, showing that, as in rhesus and humans, marmosets exhibit a \"gap effect\" - a decrease in reaction time when the fixation stimulus is removed prior to the onset of a visual saccade target. These results provide evidence that the common marmoset is a suitable model for neurophysiogical investigations of oculomotor control.\n\nNEW AND NOTEWORTHYThe ability to carry out neuronal recordings in behaving primates has provided a wealth of information regarding the neural circuits underlying the control of eye movements. Such studies require restraint of the animal within a primate chair, head fixation, methods of acclimating the animals to this restraint, and the use of operant conditioning methods for training on oculomotor tasks. In contrast to the macaque model, relatively few studies have reported in detail methods for use in the common marmoset. Here we detail custom-designed equipment and methods by which we have used to successfully train head-restrained marmosets to perform basic oculomotor tasks.

neuroscience↗