Microbial fuel cells are bio-electrochemical fuel cell systems that generate electric current using bacteria as the catalyst to oxidise organic and inorganic matter. Due to the increasing concern of climate change, they are an interesting alternative renewable energy source that are seeing a lot of focus. The aims and objectives of this book are to bring people’s attention to the existence of MFC technology, to remind them about how it works and what this technology has to offer for the future. These green machines will improve the clean-up of all the too-wet-to-burn organic wastes and will help with quick recycling of elements naturally as well as producing electricity and clean water. The book is targeted at/suitable for academics, postgraduates, late-stage undergraduates and industry workers.

Background/Objectives: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive and lethal forms of malignant neoplasm of the endocrine system, and osteopontin (OPN) has been shown to be aberrantly expressed in this tumor type. Among the five OPN splicing isoforms (OPN-SI), OPN-4 has been recently reported in several tumor types, including ATC, but its functional role(s) have not yet been elucidated. Methods: To characterize OPN-4 roles in ATC cells, OPN-4 was ectopically overexpressed in the c643 ATC cell line, generating the c643/OPN-4 cells. OPN-roles were evaluated by cell functional assays, including cell proliferation and viability, using Carboxyfluorescein Succinimidyl Ester (CFSE), crystal violet, and trypan blue assays. For migration, clonogenicity, cell cycle and apoptosis assays were used. For assessment, c643/OPN-4 cells were cultured in two-dimensional (2D) monolayers or three-dimensional (3D) spheroids with the latter being maintained in a bespoke microfluidic system. Results: OPN-4 overexpression led to a significant reduction in cell proliferation, viability, migration and clonogenicity. c643/OPN-4 cells displayed a significant accumulation in the G0/G1 phase and a decrease in the S phase of the cell cycle; however this did not affect cell death or the expression levels of other OPN-SI. In a spheroid model of c643/OPN-4 cells, no significant differences were found in spheroid size or viability when compared to those formed by control cells. Notably, OPN-4 overexpression enhanced the effects of sorafenib on cell viability under dynamic treatment conditions involving continuous perfusion. Conclusions: These early findings point to the fact that OPN-4 may reduce some aspects of tumor progression features in ATC cells and open new avenues for investigating OPN-4 as a biomarker of therapeutic response in personalized treatment strategies.

Background: Non-invasive approaches to brain tumour detection and diagnosis are limited by the absence of clinically validated circulating biomarkers. This study utilised a miniaturised tissue perfusion model to maintain human brain tumour tissue ex vivo with the aim of identifying tissue-derived proteins with potential biomarker utility. Methods: 55 tumour samples from 11 different brain tumours (glioblastoma n = 4, low-grade glioma n = 4, brain metastases n = 3) were micro-dissected and maintained ex vivo on a continuous-flow perfusion device for 168 h. Proteomic analysis of tumour effluent was performed by reversed-phase capillary liquid chromatography-mass spectrometry. Two candidate proteins—extracellular matrix protein 1 (ECM1) and cathepsin D—were quantified using ELISA. Results: All tumour subtypes retained tissue viability over 168 h of perfusion. Proteomic profiling identified 90 tissue-derived proteins in the tumour effluent. Many proteins corresponded to previously described cancer biomarkers such as glial fibrillary acidic protein (GFAP) while others, including Serpin A12 and collapsin response mediator protein-2 (CRMP2), had not yet been described in a brain tumour context. ELISA confirmed significantly higher ECM1 levels in high-grade glioma effluent compared with low-grade glioma (p = 0.0407), whereas cathepsin D levels did not differ significantly between tumour types. Conclusions: The ex vivo perfusion model effectively preserved primary and metastatic human brain tumour tissue and enabled direct characterisation of tumour-secreted proteins. The proteins identified here warrant further validation as tumour biomarkers in patient serum or cerebrospinal fluid.

Glioblastoma (GBM) is the most common and aggressive astrocytic glioma of the central nervous system with a median survival of 15 months from diagnosis. Patient outcomes have improved only marginally over the past 20 years, and GBM research is limited by models that fail to preserve the native architecture and function of tumours over time. The aim of our study was to provide a physiologically and clinically relevant microfluidic environment for the study of GBM. We designed and validated a custom-built polymethyl methacrylate (PMMA) microfluidic perfusion platform, and successfully maintained ex vivo culture of intact, fresh, patient-derived GBM biopsies for up to 12 days. Cellular viability and tissue integrity were assessed by histology, apoptosis markers, and cytokine profiling, whilst cellular stress was quantified by lactate dehydrogenase (LDH) release. LDH levels initially decreased and then remained low and stable up to 12 days. Haematoxylin and eosin staining showed that tissue architecture was preserved, with mitotic figures post-perfusion. Cleaved PARP and Annexin V immunohistochemistry revealed no significant increase in apoptosis between pre- and post-perfused tissues at 8 or 12 days. Effluent cytokine analyses demonstrated sustained secretory activity with a time-dependent modulation (across 105 analytes: p = 0.0288). Quantitative ELISAs for seven cytokines (VEGF, MMP9, CHI3L1, IL-6, IL-8, Serpin-E1, Angiopoietin-2) corroborated cytokine analysis trends and correlated strongly with array data (p < 0.001), validating assay fidelity and biological signal. Collectively, biochemical, histological and secretome readouts show that intact GBM tissues remain viable, metabolically active and analysable on this platform for up to 12 days. The system provides a practical, physiologically relevant, model for longitudinal GBM studies and lays the groundwork for scalable translational testing of patient tissue.

Early detection of hepatitis C virus (HCV) infection is crucial for eliminating this silent killer, especially in resource-limited settings. HCV core antigen (HCVcAg) represents a promising alternative to the current "gold standard" HCV RNA assays as an active viremia biomarker. Herein, a highly sensitive electrochemical magnetoimmunosensor for the HCVcAg was developed. The biosensing strategy involved capturing HCVcAg using antibody-coated magnetic beads, followed by a sandwich immunoassay before electrochemical detection on disposable screen-printed electrodes. To achieve signal amplification and consequent enhanced sensitivity, the antigen-antibody reaction was detected with a biotinylated polyclonal antibody subsequently labelled with a streptavidin poly horseradish peroxidase conjugate followed by amperometric detection via a hydroquinone/ hydrogen peroxide system. The developed biosensor exhibited a cathodic current variation directly proportional to the HCVcAg concentration over a wide range (0.1-500 ng/mL), with a detection limit of 10 pg/mL. Moreover, it successfully discriminated healthy control human plasma samples from HCVcAg-spiked samples, showed no interference from endogenous plasma constituents or cross-reactivity with other viruses tested, and possessed excellent percentage recoveries of HCVcAg (>= 92.83%), demonstrating high specificity. The proposed bioplatform remained stable for at least ten days and showed excellent clinical performance in detecting HCVcAg across a cohort of thirty-six plasma and serum samples from active hepatitis C cases, and healthy individuals, with results matching those previously obtained using clinically validated qPCR and serological testing. In summary, this biosensor provides a simple, rapid and economic alternative to other available techniques such as ELISA and qPCR for early diagnosis of HCV infection.

Tissue expressed human epidermal growth factor receptor 2 (HER2) is an established parameter of breast cancer, and it is determined in routine clinical practice by histology. This study aims to assess the prognostic value of serum HER2 protein quantified using ELISA in 66 primary breast cancer patients. The median follow-up period was 94 months. Prognostic performance was assessed through receiver operating characteristic (ROC), Cox proportional hazards regression, and Kaplan-Meier analyses. There was a significant difference between serum HER2 levels of patients with and without recurrence. Prognostic associations were significant for age, grade, nodal status, follicle-stimulating hormone, oestradiol, and serum HER2. The cut-off point for serum HER2 levels was 6.0 ng/mL. Recurrence incidence was 29% in the serum HER2high subgroup, compared to only 10% in the serum HER2low subgroup. Serum HER2 testing by ELISA holds promise as an additional tool alongside routine tissue HER2 analysis for primary breast cancer patients.

Background: Glioblastoma (GBM) remains a highly aggressive and treatment-resistant brain tumour, characterized by pro- found immune suppression. Chitinase 3-like 1 (CHI3L1), a cytokine implicated in tumour progression and im- mune modulation, has been associated with poor prognosis in GBM. This study examines CHI3L1 expression in ex vivoGBM tissue maintained on a microfluidic platform, investigating changes on application of standard-of- care chemotherapy. To assess CHI3L1 secretion in patient-derived GBM tissue under physiological perfusion conditions and deter- mine its response to clinically relevant chemotherapeutic agents. GBM biopsy samples from 13 patients were cultured on a microfluidic perfusion system for 8-12 days. Tissue was either maintained untreated or exposed to a combination of Temozolomide (TMZ) and a PRMT inhibitor. CHI3L1 levels were quantified using a Proteome Profiler Human XL Cytokine Array and validated via ELISA. The persistence and upregulation of CHI3L1 in treated GBM tissue highlights a potential role in immune eva- sion and/or therapeutic resistance. These findings suggest that targeting CHI3L1 could enhance the efficacy of current GBM treatments and improve patient outcomes; further clinical investigations are required.

Conventional techniques for treating wastewater consume significant energy and depending on effectiveness, may result in secondary contamination. In this regard, the microbial fuel cell (MFC) technology has shown much promise as a revolutionary wastewater treatment + energy generation hybrid. This is due to the unique ability of electroactive organisms to generate direct electricity, recovering electrons from the breakdown and consumption of organic compounds in wastewater. This article critically assesses the current development of MFC technology, particularly in the last two years, focussing on the technology's economic and environmental feasibility. Even though there is a significant body of literature on MFCs with continuously increasing performance levels, the technology has not yet got fully commercialised to become part of urban planning or energy policy; this implies a lack of government consideration as a result of the absence of industrial scale research. The article presents the case for MFCs from a technology readiness level and life cycle assessment perspectives and explains why it is still premature to draw conclusions based on these two metrics.

Incorporating precision oncology into cancer management has begun to improve clinical outcomes. Accurate sampling techniques that detect molecular aberrations are crucial for effective implementation. Circulating tumor cells (CTCs), derived from primary or metastatic sites and present in the blood, are proposed as useful diagnostic tools, though their use has been limited due to their rarity, especially in early-stage cancers. This study presents a novel immunomagnetic microfluidic device that efficiently isolates CTCs for analyzing epidermal growth factor receptor (EGFR) mutations in patients with non-small cell lung cancer (NSCLC). The device was designed and laser-cut from polymethylmethacrylate. Validation experiments involved spiking PC-9 cells (an established lung cancer cell line containing GLU 746-ALA 750 deletion mutations in exon 19 of the EGFR gene) into media and isolating these cells. Exons 18 – 21 of EGFR were amplified using a polymerase chain reaction to demonstrate the device’s rapid mutation detection capability. Next-generation sequencing was used to characterize these exons in a cohort of 38 NSCLC patients, successfully isolating CTCs from all. Among these patients, 30 (79%) had EGFR mutations, with exon 19 showing the highest mutation rate (87%) and exon 21 the highest point mutation rate (23%). Our device captured CTCs effectively in <1 h, enabling mutation detection. Further studies are needed to assess the prognostic significance of these mutations, but this technology has potential applications in various solid tumors.

The application of microbial fuel cells in sanitation has demonstrated feasibility in supplying electricity and providing safety in underserved communities, especially at toilet blocks. Two different designs of urine fed MFC cascades, ceramic MFCs (c-MFC) and self-stratifying MFCs (s-MFC), have been employed in large-scale feasibility studies. As part of a pre-commercialisation approach, this study verified the resilience of each design when a commercial disinfectant was introduced into the system. Five different conditions, varying in concentrations (24.2 mM-604.5 mM) and the total volume (50-500 mL) of sodium hypochlorite disinfectant introduced, were tested. Upon adding the disinfectant, both types of MFC-cascades exhibited rapid power drops with response times lower than 5 min in all tested conditions, followed by relatively swift recovery times of up to 250 min. The volume of disinfectant introduced had a greater impact on power output than its concentration or dose. Comparing the two designs, the c-MFC demonstrated a much larger voltage drop, up to 0 mV, and shorter recovery time compared to the s-MFC under most test conditions, mainly attributed to the presence (c-MFC) or absence (s-MFC) of a membrane. Overall, both types of MFCs exhibited strong resilience to sodium hypochlorite additions, thereby highlighting the commercial potential of the technology towards safe off-grid sanitation.

Abstract AIMS The blood brain barrier (BBB) presents one of the main obstacles for the development of novel glioblastoma treatments. The various transporters and enzymes within the BBB reduce the disposition of novel therapies into the brain. There is a need for an in vitro BBB-glioma model with proven efficacy of predicting CNS delivery at earlier stages in the drug discovery pipeline. METHOD A BBB-glioma lab-on-a-chip model could enable rapid identification of novel therapies that can cross the BBB at earlier stages of the drug development process, before going to clinical trials. The application of microfluidic technology allows for the detection of soluble tissue-derived factors and the assessment of the levels of soluble markers released from malignant tissue. Adding ‘flow’ to the model, is more reflective of the dynamic flow present at the BBB. RESULTS Validation studies of the current BBB model and converting this model over to a lab-on-a-chip model have shown the presence of an endothelial cell barrier and the tight junction proteins Zo-1 and Claudin-1, through western blotting, immunocytochemistry, and permeability experiments using varying molecular weights of FitC-dextran. These validation studies have been completed using brain endothelial monolayers on polycarbonate transwell inserts, with the immortalised cell line HCMEC/d3, and the short-term culture cells HBMEC. Also, validation studies using a co-culture of endothelial cells, astrocytes and pericytes have begun, using the same techniques to show a barrier has formed, on both the current BBB model and the lab-on-a-chip model. CONCLUSION The aim of this study is to combine the use of an all-human BBB-glioma model with lab-on-a-chip technology to produce a high throughput model to test novel therapies at an earlier stage. The lab-on-a-chip model will be validated for the purpose of measuring permeability and drug response and to investigate the expression of novel targets verses expression in conventional models.

Abstract AIMS Glioblastoma (GBM) is the deadliest and most common form of brain tumour. The average length of survival for GBM patients remains low, in part, due to a lack of targeted therapeutics. Identification and validation of novel therapeutic targets is therefore crucial. Chomobox 2 (CBX2; a component of polycomb repressive complex 1 (PRC1)), represses gene expression and has a pro-oncogenic role in several aggressive cancer subtypes. CBX2 expression is elevated in GBM, however, very little is known about its role in this deadly disease. We aim to assess the phenotypic and gene expression effects of preventing CBX2-chromatin interaction in patient-derived GBM cell models, and to determine the functional mechanisms by which CBX2 promotes GBM progression; thereby determining its potential as a novel therapeutic target. METHOD Crucial to impactful target validation is the use of translationally relevant models, this study utilises a 3D spheroid model, using patient-derived cells, which more closely recapitulates the in vivo tumour microenvironment. GBM cell monolayers and spheroids were treated with SW2_152F, a selective CBX2 inhibitor. The phenotypic effects of CBX2 inhibition were assessed by cell count, CellTiter-Glo viability assays, fluorescence microscopy, and flow cytometry. Genes and biological pathways differentially regulated following CBX2 inhibition were assessed by RNA-sequencing and Gene Set Enrichment Analysis (GSEA). RESULTS Inhibition of CBX2 induced cell death and reduced cell viability in both 2D and 3D GBM cultures. RNA-seq and GSEA of SW2_152F-treated cells identified dysregulation of gene signatures involved in pro-oncogenic MYC and E2F signalling pathways, and dysregulation of genes associated with G2/M cell cycle progression. CONCLUSION Preventing CBX2-chromatin interaction induces GBM cell death. Further research will identify CBX2- chromatin binding sites to characterise the activity and direct regulatory role of CBX2 in GBM. Furthermore, we will assess the phenotypic and gene expression effects of CBX2 inhibition and depletion on ex vivo maintained GBM tissue biopsies.

The death of dopamine-producing neurons in the substantia nigra in the base of the brain is a defining pathological feature in the development of Parkinson's disease (PD). PD is, however, a multi-systemic disease, also affecting the peripheral nervous system and gastrointestinal tract (GIT) that interact via the gut-brain axis (GBA). Our dual-flow GIT-brain microphysiological system (MPS) was modified to investigate the gut-to-brain translocation of the neurotoxin trigger of PD, 1-methyl-4-phenylpyridinium (MPP+), and its impact on key GIT and brain cells that contribute to the GBA. The modular GIT-brain MPS in combination with quantitative and morphometric image analysis methods reproduces cell specific neurotoxin-induced dopaminergic cytotoxicity and mitochondria-toxicity with the drug having no detrimental impact on the viability or integrity of cellular membranes of GIT-derived colonic epithelial cells. Our findings demonstrate the utility and capability of the GIT-brain MPS for measuring neuronal responses and its suitability for identifying compounds or molecules produced in the GIT that can exacerbate or protect against neuronal inflammation and cell death. (c) 2024 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International (CC BY-NC-ND) license (

Advancements in 3-Dimensional (3D) culture models for studying disease have increased significantly over the last two decades, but fully understanding how these models represent in vivo still requires further investigation. The current study investigated differences in gene expression between a baseline sample and that maintained on a tissue-on-chip perfusion device for up to 96 h, with and without clinically-relevant doses of irradiation, to allow differentiation of model and treatment effects. Tumour tissue samples from 7 Head and Neck Squamous Cell Carcinomas (HNSCC) patients were sub-divided and either fixed immediately upon excision or maintained in a tissue-on-chip device for 48 and 96 h, with or without 2 Gray (Gy) or 10 Gy irradiation. Gene expression was measured using an nCounter® PanCancer Progression Panel. Differentially expressed genes between pre- and post-ex vivo culture, and control and irradiated samples were identified using nSolver software (version 4.0). The secretome from the tumour-on-chip was analysed for the presence of cytokines using a Proteome Profiler™ platform. Significant numbers of genes both increased (n = 6 and 64) and decreased (n = 18 and 58) in expression in the tissue maintained on-chip for 48 and 96 h, respectively, compared to fresh tissue; however, the irradiation schedule chosen did not induce significant changes in gene expression or cytokine secretion. Although HNSCC tissue maintained ex vivo shows a decrease in a large proportion of altered genes, 25% and 53% (48 and 96 h) do show increased expression, suggesting that the tissue remains functional. Irradiation of tumour tissue-on-chip needs to be conducted for longer time periods for specific gene changes to be observed, but we have shown, for the first time, the feasibility of using this perfusion platform for studying the genomic response of HNSCC tissue biopsies.

Glioblastoma (GBM) is a deadly disease with a poor prognosis, there is therefore a crucial need for novel therapeutic targets. Current preclinical models of GBM fail to predict clinical outcomes, thus, new translationally relevant models are urgently needed for reliable therapeutic target validation. 3D spheroid culture of cancer cells has been shown to better reflect tumour biology than 2D monolayer culture, as has culturing cells in flow-based microfluidic devices, which mimic key aspects of the tumour microenvironment. Gene knockdown by siRNA is a key preclinical target validation tool, however, siRNA-mediated knockdown of cancer spheroids in microfluidic culture has not yet been demonstrated. Here we describe a simple and robust microfluidic device that can maintain GBM spheroids (U87 cells) for at least 7 days. Via RNA sequencing analysis, we demonstrate that spheroids grown in microfluidic culture are more proliferative than spheroids grown in static plate culture and downregulate genes associated with cell adhesion, potentially offering insights into the metastatic process. Comparison of target gene (PRMT2 and RAB21) knockdown using siRNA between 2D monolayer cultured cells, static spheroid culture and spheroids maintained in the microfluidic device showed that gene expression (as measured by quantitative-PCR) was significantly reduced in all culture systems. Knockdown was most efficient in cells grown in 2D monolayer culture followed by static spheroid culture, but we also demonstrate [Formula: see text] knockdown efficiency using the microfluidic device. In summary, this study describes an easy-to-use microfluidic culture platform and provides evidence that pre-clinical siRNA-mediated target validation studies will be possible in flow systems that mimic tumour physiology.

BACKGROUND:Vascular endothelial growth factor (VEGF) -A and -C act as multifunctional molecules and growth factors, while VE-cadherin (cadherin 5, CDH5) is the endothelial junction protein. AIM:To assess the relationship between intratumoral VEGF -A, -C and CDH5 levels and clinical outcome, in primary, early-stage, breast cancer patients. PATIENTS AND METHODS:The study included 69 node-negative (N0) breast cancer patients, all of whom had not received any prior hormonal or chemotherapeutic systemic therapy that would affect the course of disease. The median follow-up period was 144 months. Intratumoral mRNA levels of VEGF -A, -C and CDH5 were determined by RT-qPCR. Prognostic performance was evaluated by Cox proportional hazards regression, Kaplan-Meier analysis, as well as by the multivariable approach based on the least absolute shrinkage and selection operator (LASSO) logit regression. Classification of patients into the low and high subgroups was performed using the outcome-oriented cut-off point categorization approach. RESULTS:Of the measured mRNAs, only CDH5 mRNA (t = -2.17; p = 0.04) and VEGF-C mRNA (t = -2.41; p = 0.03) showed significant differences between values in patient subgroups with distant metastasis and those without recurrences, respectively. These t-test results were in agreement with the Cox regression by which CDH5 mRNA reached the most pronounced hazard ratio (HR=2.07; p = 0.05), followed by VEGF-C mRNA (HR=1.59; p = 0.005). HR values above 1.0 indicate that high levels of either CDH5 or VEGF-C mRNAs associated with a higher risk of poor clinical outcome. Distant recurrence incidence was 26% for the CDH5high and 3% for the CDH5low subgroup (Kaplan-Meier analysis). Distant recurrence incidence was 23% for the VEGF-Chigh and 0% for VEGF-Clow subgroup. The independent prognostic value of VEGF-C mRNA was confirmed by LASSO regression. CONCLUSION:Intratumoral VEGF-A levels did not associate with disease outcome in primary, early-stage, breast cancer patients, whilst raised levels of either CDH5 or VEGF-C prognosticated a high risk of distant metastasis.

Introduction. Clear cell renal cell carcinoma (ccRCC) is a highly vascularized and clinically aggressive cancer. In the UK, there are around 9000 diagnoses and 3000 deaths annually. Targeted therapies that inhibit angiogenic regulators, such as bevacizumab (monoclonal antibody which inhibits vascular endothelial growth factor A, VEGF-A), have limited efficacy due to drug resistance. Expression of calcitonin receptor-like receptor (CLR) is upregulated in ccRCC and correlates with disease outcome. The expression of adrenomedullin (AM), peptide agonist of CLR and angiogenic factor, is upregulated in ccRCC. We hypothesized that the CLR signaling axis acts as an alternative pathway that enables resistance to drugs targeting the VEGF-A pathway in ccRCC. In the present study, we aimed to determine whether this signaling axis remains active in VEGF-A-stimulated and bevacizumab-treated human endothelial cells. Methods. Primary human dermal blood vessel endothelial cells (HDBEC; PromoCell) were characterized by immunofluorescence utilizing pan-endothelial (cluster of differentiation 31 and 144, CD31 and CD144, and von Willebrand factor, vWF) and lymphatic-specific (lymphatic vessel endothelial hyaluronan receptor 1, LYVE-1, and prospero homeobox 1, PROX1) markers and confocal microscopy. The activities of CLR agonists (AM; intermedin, IMD; calcitonin gene-related peptide, CGRP; 10−12 - 10−6 M at 10 min) and VEGF-A (50 ng/ml over 30-minute time course) were measured by analyzing phosphorylation of p44/42 mitogen-activated protein kinases (MAPK) using immunoblotting. HDBEC were pre-treated with bevacizumab or human IgG1 at 250 ng/ml and then stimulated with individual CLR agonists at 10−6 M for 10 minutes. Experiments were repeated at least three times. Data were analyzed using Shapiro-Wilk normality, unpaired statistics and appropriate multiple comparisons tests utilizing GraphPad Prism and p<0.05 interpreted as significant. Results. HDBEC were a pure population. AM, IMD and CGRP induced p44/42 MAPK phosphorylation at 10−6 M (p<0.05). For all three CLR agonists and VEGF-A, p44/42 MAPK phosphorylation peaked after 10 minutes of incubation (p<0.05). Bevacizumab treatment at 250 ng/ml was sufficient to inhibit VEGF-A-induced p44/42 MAPK phosphorylation (p<0.05). AM, IMD and CGRP were all able to induce phosphorylation of p44/42 MAPK after bevacizumab treatment (p<0.01). Conclusions. The findings that CLR agonists induce p44/42 MAPK phosphorylation in VEGF-A-stimulated HDBEC after bevacizumab treatment support our hypothesis. This work provides a foundation for investigating the role of the CLR signaling axis in mechanisms of ccRCC resistance to drugs targeting the VEGF-A pathway. Citation Format: Matthew A. Morfitt, John Greenman, Anthony Maraveyas, Leonid L. Nikitenko. Calcitonin receptor-like receptor agonists induce p44/42 MAPK phosphorylation in VEGF-A-stimulated human blood endothelial cells after bevacizumab treatment [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B001.

Abstract AIMS • Assess/evaluate transcriptomic changes in GBM, maintained on a microfluidics system, in response to treatment with arginine methylation inhibitor GSK3368715, currently in clinical trials, to identify novel therapeutic strategies, synergising with personalised patient care and precision medicine • Investigate molecular differences between healthy brain and GBM maintained on-chip in response to GSK3368715, including proliferation, apoptosis and splicing. METHOD GBM biopsies from Hull Royal Infirmary, or healthy mouse brain tissues, were maintained on-chip for 8- days and perfused with GSK3368715-treated media, at 3 μl/min, mimicking the in vivo environment. RNA- sequencing determined thousands of differentially expressed genes and alternative splicing (AS) events resulting from GSK3368715 treatment. Immunohistochemistry displayed the effect of GSK3368715 on healthy mouse brain tissue to ascertain the specificity of the arginine methylation inhibitor in leading to GBM apoptosis. RESULTS RNA-sequencing showed highly significant GO-term-enrichment in ribosome and translation pathways, suggesting decreased protein synthesis capacity after GSK3368715 treatment. Additionally, several hundreds of genes were found to be undergoing AS, compatible with a mechanism where changes in the arginine methylation pattern of spliceosome member FUS, upon GSK3368715 treatment, contribute to AS. Of these events, 136 (more than expected by chance, χ2 = 7.16, p = 0.007) were known FUS splicing targets. GO-term-enrichment of these AS events was found in DNA damage and cell death . Together these results are consistent with immunohistological findings that GSK3368715 causes apoptosis in GBM-on-chip tissue, in a specific manner. CONCLUSIONS Our results highlight an exciting potential therapeutic target for GBM, via induction of alternative splicing path- ways, through arginine methylation inhibition with GSK3368715.

Introduction. Clear cell renal cell carcinoma (ccRCC) is a highly vascularized and clinically aggressive kidney cancer, with high rates of metastasis and recurrence after cytoreductive nephrectomy. ccRCC can be treated with anti-angiogenic drugs, however drug resistance usually develops within one year and 5-year survival for metastatic cases is approximately 10%. Calcitonin receptor-like receptor (CLR) is a G-protein-coupled receptor (GPCR) expressed in endothelial cells (EC) in core- (intracellular) and terminally-glycosylated (cell surface-associated) forms. CLR and its three agonists play a role in angiogenesis. In ccRCC, CLR is upregulated in tumor vessels when compared to vessels in adjacent tissues. The aims of this study were to: (1) investigate which types of vessels (blood or lymphatic) in ccRCC tissues express CLR and (2) determine whether ccRCC tumor cells affect expression of this GPCR in EC. Methods. CLR expression was analyzed in sections of ccRCC tissues (primary tumors) from three patients by using immunohistochemistry and our rabbit anti-human CLR antibody LN1436, with controls performed using the antigen (21 amino-acid peptide). Distribution of CLR immunoreactivity was compared to pan-EC (cluster of differentiation 31, CD31) and lymphatic EC-specific (podoplanin, PDPN) marker expression patterns, revealed by using immunohistochemistry on serial sections with mouse monoclonal antibodies JC/70A and D2-40 respectively. Two ccRCC cell lines (786-O and KTCTL-140) were co-cultured with human dermal blood vessel EC (HDBEC) in vitro for 48 hours. Cell types were separated by using magnetic-activated cell sorting (MACS) with anti-CD31 antibody-conjugated microbeads. CLR expression in ccRCC and HDBEC before, during and after co-culture was analyzed by using immunoblotting and LN1436 antibody. Results. In ccRCC tissue sections, CLR immunoreactivity was detected predominantly in blood and not lymphatic vessels. Core- and terminally-glycosylated forms of CLR were expressed in HDBEC, and the expression levels of both were upregulated after co-culture with each of the two studied ccRCC cell lines. Conclusions. These data advance knowledge about CLR expression in ccRCC and suggest that tumor cells may be responsible for its upregulation in EC within the tumor microenvironment. Our findings warrant further investigation of the potential role of the expressed in tumor blood vessels GPCR CLR as a prognostic marker and/or a target for therapy in ccRCC. Citation Format: Matthew A. Morfitt, John Greenman, Anthony Maraveyas, Adrian L. Harris, Leonid L. Nikitenko. Calcitonin receptor-like receptor is expressed in blood vessels in clear cell renal cell carcinoma and upregulated in endothelial cells co-cultured with tumor cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A020.