Cell competition is an emerging mechanism in which mammalian tissues maintain homeostasis by eliminating less fit (loser) cells through direct interactions with fitter (winner) neighbouring cells. In cancer, these competitive interactions may drive tumour evolution; however, spatial organisation and clinical relevance of these events remain poorly understood. One mechanism by which winner cells eliminate loser cells is engulfment, resulting in cell-in-cell (CIC) formation. Although CICs have been observed in many tumour types for over a century, their cellular composition, spatial context, interactions with the tumour microenvironment, and biological significance in human cancers remain unclear. Here, we systematically characterised the cellular identity and functional states of CICs in situ, examined their spatial interactions within the tumour microenvironment, and assessed their clinical relevance using spatially resolved single-cell data from a large cohort of colorectal cancer patients. We demonstrated that CICs occurred predominantly between cancer cells but also involved cancer stem cell (CSC)-like populations and cytotoxic T cells. Engulfed (inner) cancer and CSC-like cells displayed molecular features consistent with a loser-cell phenotype, including increased apoptosis and reduced proliferation, whereas outer cancer cells exhibited winner-cell features such as upregulated glycolysis. Live-cell time-lapse experiments demonstrated that glucose accumulated in inner cells during lysosomal degradation following cell engulfment. Spatial analysis further revealed distinct CIC neighbourhoods, which we defined based on proximity to engulfment events. Cells within these regions, particularly CSC-like cells and cytotoxic T cells, exhibited increased metabolic stress, suggesting local competition for nutrients. Importantly, the presence of cytotoxic T cells within CIC neighbourhoods and spatial co-occurrence between cancer cells and CSC-like populations were associated with improved patient outcomes. Together, our findings demonstrate that cell engulfment defines spatially organised competitive niches and may reflect cell competition within complex tumour microenvironments.

We describe a reproducible 3D migration assay to model the migratory and invasive potential of patient-derived glioblastoma gliomaspheres from newly diagnosed and recurrent tumors under clinically relevant hypoxic conditions. Uniform sized gliomaspheres are transferred onto a thin layer of extracellular matrix and co-cultured with magnetic iron oxide nanoparticles, which enable their centralized localization within culture wells via placement onto a magnetic plate holder. The inclusion of magnetic iron oxide nanoparticles on seeding facilitates precise, localized imaging of individual patient derived gliomaspheres via a gentle magnetic force and optimizes automated image processing pipelines by reducing positional variability. This assay supports detailed study of glioblastoma migratory behavior in a physiologically relevant microenvironment and allows direct comparison of invasive potential and migratory behavior between newly diagnosed and recurrent patient derived gliomaspheres. The method is compatible with live cell imaging and multiplexed analysis, offering a scalable platform for preclinical investigation of glioblastoma migration, invasive potential, and therapeutic response.

Immune-based strategies have so far failed to demonstrate clinical benefit in glioblastoma (GBM), largely due to the profound immunosuppressive tumor microenvironment (TME). To achieve more predictive preclinical insights, advanced in vivo models that faithfully recapitulate the human brain immune landscape are urgently needed. Here, we established GBM patient-derived orthotopic xenografts (PDOXs) across diverse mouse strains, including humanized models. Humanization was achieved through transplantation of CD34⁺ hematopoietic stem cells (HU-CD34⁺) or peripheral blood mononuclear cells (HU-PBMC). Both models successfully reconstituted human T-cells systemically, with stronger engraftment in HU-CD34⁺ mice. We observed selective infiltration and spatial organization to intracranial GBM tumors, including exhausted, memory-like, and regulatory CD4⁺ T-cell phenotypes, TIM-3⁺ immunosuppressive-like myeloid cells and intratumoral B cells. Mouse microglia-derived tumor-associated macrophages (TAMs) remained the dominant immunosuppressive immune population. Anti-PD-1 therapy, but not anti-GITR, modestly modulated the infiltration dynamics, demonstrating the susceptibility of the reconstructed adaptive immunity to immunotherapeutic intervention. These findings position humanized GBM PDOXs as a relevant preclinical platform to interrogate tumor-immune interactions and evaluate immunotherapeutic strategies in a human context. ### Competing Interest Statement The authors have declared no competing interest. Fonds National de la Recherche, https://ror.org/039z13y21, PRIDE19/14254520/i2TRON, C20/BM/14646004/GLASS-LUX, C21/BM/15739125/DIOMEDES, INTER/GACR/23/18089030-MITOFIT, C20/BM/14582635, C20/BM/14592342, C20/BM/14592342 ERA-NET TRANSCAN-3, FNR INTER/TRANSCAN22/17612718/PLASTIG, Research Ireland 21/RI/9787 Télévie-FNRS, ImmoGBM n° 7.8505.20/7.6603.22 Luxembourg Institute of Health, https://ror.org/012m8gv78

5-15% of all colorectal cancers (CRCs) are mucinous. Mucinous CRCs are associated with an inhibited response to standard adjuvant and neoadjuvant therapies. Serine-Arginine Protein Kinase 1 (SRPK1) is an enzyme, which modulates the activity of multiple splicing factors. SRPK1 under-expression is associated with resistance to platinum-based chemotherapeutic agents in multiple tumor types. The objectives of this study were to evaluate SRPK1 expression in mucinous CRC and to explore the potential relationship between differential SRPK1 expression and oxaliplatin resistance in mucinous CRC. Rectal cancer and CRC Tissue Microarrays (TMA) were stained with SRPK1 to compare expression between mucinous and non-mucinous tumors. SRPK1 expression was analyzed in mucinous and non-mucinous CRC cell lines. Cells were treated with oxaliplatin to explore differences in treatment response. Mucinous cells were transfected with an SRPK1 CRISPR/Cas9 lentiviral activation plasmid to investigate the relationship between SRPK1 expression and oxaliplatin resistance. The TMA cohorts included 117 patients with mucinous and 441 patients with non-mucinous CRC. SRPK1 was found to be under-expressed in both the mucinous rectal cancer (P < 0.001) and CRC cohorts (P = 0.003). On univariate analysis, SRPK1 under-expression was found to be associated with worse 5-year OS (P = 0.001). Treatment of mucinous CRC cells with oxaliplatin did not result in a significant increase in cell death (P = 0.149). However overexpression of SRPK1 following transfection with a CRISPR/CAS9 activation plasmid resulted in a significant increase in sensitivity of these cells to oxaliplatin treatment (P = 0.029). SRPK1 is under-expressed in mucinous CRC, and under-expression is associated with worse OS. This may be due to the positive effects of SRPK1 on oxaliplatin sensitivity.

Previous research provided evidence pointing towards an important role for bacterial infection with Fusobacterium nucleatum (F. nucleatum) and F. animalis (previuosly F. nucleatum subsp. animalis) in colorectal cancer (CRC) progression. Both are oral commensal Gram negative anaerobes with recent evidence suggesting F. animalis predominates in CRC (Zepeda-Rivera et al., 2024). We recently applied spatial transcriptomics to demonstrate local effects of Fusobacterium infection on cancer and immune cell gene expression in CRC (Duggan et al, Gut Microbes, 2024). While Fusobacterium infection was observed in specific patches in the tumors, the precise cell types infected with bacteria and the precise localization of bacteria in cells still requires further elucidation. Commercially available strains of F. nucleatum (ATCC 25586) and F. animalis (ATCC 51191) were purchased and separately co-cultured with MDA-MB-468 breast cancer cells. Immuno cytochemistry using a commercial Fusobacteria antibody (ANT0084) and a Fusobacteria antiserum (Prof Slade group), along with pancytokeratin and Hoechst staining was undertaken. High resolution confocal microscopy using an LSM 980 Airyscan 2 microscope (Carl Zeiss, Jena, Germany) was performed to create image stacks of optical sections. CRC tissue sections were subsequently prepared and stained by immunohistochemistry (IHC) for pancytokeratin, CD68, and Fusobacteria (Slade antisera). Imaging of the full tumor face was undertaken at a lower resolution using Cell DIVE (Leica Microsystems, Wetzlar, Germany) to select regions rich in Fusobacteria for further study. Selected regions were then imaged using high resolution confocal microscopy. Fusobacteria antisera stained both F. nucleatum and F. animalis while the commercially produced antibody stained only F. nucleatum. Both bacteria can populate the cell surroundings, cell surface, cytoplasm as well as nucleus. The typical morphology of both F. nucleatum and F. animalis is seen, allowing antibody validation. In CRC tissue intracellular bacteria are seen in both pancytokeratin positive tumor epithelial cells as well as in CD68 (Cluster of Differentiation 68) positive cells (tumor associated macrophages). Tumor associated macrophages (TAMs) have an important role in the tumor immmune microenvironment. In particular, studies have highlighted the importance of M1/M2 macrophage polarization on immune-evasion and prognosis. Our spatial transcriptomic study highlighted reduced M2 polarization as associated with increased Fusobacterial load. The intracellular location of Fusobacteria may suggest a mechanism for altered macrophage polarization. References: Zepeda-Rivera et al, Nature Vol 628, pages 424-432 (2024)Flanagan et al, Eur J Clin Microbiol Infect Dis. 2014 Aug;33(8):1381-90 Duggan et al, Gut Microbes. 2024 Jan-Dec;16(1):2350149. Heiko Dussmann, Barry Maguire, Caoimbhe Burke, Arman Raman, John Burke, William M. Gallagher, Jochen H. Prehn. High resolution analysis of Fusobacterium infection in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6531.

Cell-in-cell (CIC) structures, in which one cell is entirely engulfed by another, have been associated with poor outcomes in cancers. However, the mechanisms underlying this association remain poorly understood. We performed multiplex imaging of 56 cell identity, cell ‘state’ and cancer ‘hallmark’ proteins to characterise CICs, map their spatial interactions, and assess clinical associations across 444 tumour cores from 148 colorectal cancer patients, which contained over one million spatially resolved cells. We found that tumour regions containing CICs were associated with lower levels of cytotoxic T cells. We identified upregulated glucose metabolism as a consistent metabolic hallmark of CICs independent of cell type. Spatial analyses revealed that T cells adjacent to CICs underwent selective remodeling with distinct apoptotic and metabolic signatures. Finally, the presence of T cells within CIC neighbourhoods identified a subset of patients with improved survival. Our findings suggest that CICs may be a feature of metabolically competitive niches and a potential factor contributing to T-cell exclusion in tumours.

Mucinous colorectal cancer (CRC) is a common histological subtype of colorectal adenocarcinoma, associated with a poor response to chemoradiotherapy. The commensal facultative anaerobes fusobacteria, have been associated with poor prognosis specifically in mesenchymal CRC. Interestingly, fusobacterial infection is especially prevalent in mucinous CRC. The objective of this study was therefore to increase our understanding of beneficial and detrimental effects of fusobacterial infection, by contrasting host cell signaling and immune responses in areas of high vs. low infection, using mucinous rectal cancer as a clinically relevant example. We employed spatial transcriptomic profiling of 106 regions of interest from 8 mucinous rectal cancer samples to study gene expression in the epithelial and immune segments across regions of high versus low fusobacterial infection. Fusobacteria high regions were associated with increased oxidative stress, DNA damage, and P53 signaling. Meanwhile regions of low fusobacterial prevalence were characterized by elevated JAK-STAT, Il-17, Il-1, chemokine and TNF signaling. Immune masks within fusobacterial high regions were characterized by elevated proportions of cytotoxic (CD8+) T cells (p = 0.037), natural killer (NK) cells (p < 0.001), B-cells (p < 0.001), and gamma delta T cells (p = 0.003). Meanwhile, fusobacteria low regions were associated with significantly greater M2 macrophage (p < 0.001), fibroblast (p < 0.001), pericyte (p = 0.002), and endothelial (p < 0.001) counts.

Colorectal cancer (CRC) is one of the most frequently occurring cancers, but prognostic biomarkers identifying patients at risk of recurrence are still lacking. In this study, we aimed to investigate in more detail the spatial relationship between intratumoural T cells, cancer cells, and cancer cell hallmarks as prognostic biomarkers in stage III colorectal cancer patients. We conducted multiplexed imaging of 56 protein markers at single-cell resolution on resected fixed tissue from stage III CRC patients who received adjuvant 5-fluorouracil (5FU)-based chemotherapy. Images underwent segmentation for tumour, stroma, and immune cells, and cancer cell 'state' protein marker expression was quantified at a cellular level. We developed a Python package for estimation of spatial proximity, nearest neighbour analysis focusing on cancer cell-T-cell interactions at single-cell level. In our discovery cohort (Memorial Sloan Kettering samples), we processed 462 core samples (total number of cells: 1,669,228) from 221 adjuvant 5FU-treated stage III patients. The validation cohort (Huntsville Clearview Cancer Center samples) consisted of 272 samples (total number of cells: 853,398) from 98 stage III CRC patients. While there were trends for an association between the percentage of cytotoxic T cells (across the whole cancer core), it did not reach significance (discovery cohort: p = 0.07; validation cohort: p = 0.19). We next utilised our region-based nearest neighbour approach to determine the spatial relationships between cytotoxic T cells, helper T cells, and cancer cell clusters. In both cohorts, we found that shorter distance between cytotoxic T cells, T helper cells, and cancer cells was significantly associated with increased disease-free survival. An unsupervised trained model that clustered patients based on the median distance between immune cells and cancer cells, as well as protein expression profiles, successfully classified patients into low-risk and high-risk groups (discovery cohort: p = 0.01; validation cohort: p = 0.003). (c) 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The ketogenic diet is an emerging therapeutic approach for refractory epilepsy, as well as certain rare and neurodegenerative disorders. The main ketone body, β-hydroxybutyrate (BHB), is the primary energy substrate endogenously produced in a ketogenic diet, however, mechanisms of its therapeutic actions remain unknown. Here, we studied the effects of BHB on mitochondrial energetics, both in non-stimulated conditions and during glutamate-mediated hyperexcitation. We found that glutamate-induced hyperexcitation stimulated mitochondrial respiration in cultured cortical neurons, and that this response was greater in cultures supplemented with BHB than with glucose. BHB enabled a stronger and more sustained maximal uncoupled respiration, indicating that BHB enables neurons to respond more efficiently to increased energy demands such as induced during hyperexcitation. We found that cytosolic Ca 2+ was required for BHB-mediated enhancement of mitochondrial function, and that this enhancement was independent of the mitochondrial glutamate-aspartate carrier, Aralar/AGC1. Our results suggest that BHB exerts its protective effects against hyperexcitation by enhancing mitochondrial function through a Ca 2+ -dependent, but Aralar/AGC1-independent stimulation of mitochondrial respiration.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two aging-related neurodegenerative diseases that share common key features, including aggregation of pathogenic proteins, dysfunction of mitochondria, and impairment of autophagy. Mutations in ubiquilin 2 (UBQLN2), a shuttle protein in the ubiquitin-proteasome system (UPS), can cause ALS/FTD, but the mechanism underlying UBQLN2-mediated pathogenesis is still uncertain. Recent studies indicate that mitophagy, a selective form of autophagy which is crucial for mitochondrial quality control, is tightly associated with neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, and ALS. In this study, we show that after Parkin-dependent ubiquitination of damaged mitochondria, UBQLN2 is recruited to poly-ubiquitinated mitochondria through the UBA domain. UBQLN2 cooperates with the chaperone HSP70 to promote UPS-driven degradation of outer mitochondrial membrane (OMM) proteins. The resulting rupture of the OMM triggers the autophagosomal recognition of the inner mitochondrial membrane receptor PHB2. UBQLN2 is required for Parkin-mediated mitophagy and neuronal survival upon mitochondrial damage, and the ALS/FTD pathogenic mutations in UBQLN2 impair mitophagy in primary cultured neurons. Taken together, our findings link dysfunctional mitophagy to UBQLN2-mediated neurodegeneration.

Glioblastoma (GBM) is an aggressive brain cancer that typically results in death in the first 15 months after diagnosis. There have been limited advances in finding new treatments for GBM. In this study, we investigated molecular differences between patients with extremely short (≤ 9 months, Short term survivors, STS) and long survival (≥ 36 months, Long term survivors, LTS).Patients were selected from an in-house cohort (GLIOTRAIN-cohort), using defined inclusion criteria (Karnofsky score > 70; age < 70 years old; Stupp protocol as first line treatment, IDH wild type), and a multi-omic analysis of LTS and STS GBM samples was performed.Transcriptomic analysis of tumour samples identified cilium gene signatures as enriched in LTS. Moreover, Immunohistochemical analysis confirmed the presence of cilia in the tumours of LTS. Notably, reverse phase protein array analysis (RPPA) demonstrated increased phosphorylated GAB1 (Y627), SRC (Y527), BCL2 (S70) and RAF (S338) protein expression in STS compared to LTS. Next, we identified 25 unique master regulators (MR) and 13 transcription factors (TFs) belonging to ontologies of integrin signalling and cell cycle to be upregulated in STS.Overall, comparison of STS and LTS GBM patients, identifies novel biomarkers and potential actionable therapeutic targets for the management of GBM.

There is currently an urgent need to identify factors predictive of immunogenicity in colorectal cancer (CRC). Mucinous CRC is a distinct histological subtype of CRC, associated with a poor response to chemotherapy. Recent evidence suggests the commensal facultative anaerobe Fusobacterium may be especially prevalent in mucinous CRC. The objectives of this study were to assess the association of Fusobacterium abundance with immune cell composition and prognosis in mucinous CRC. Our study included two independent colorectal cancer patient cohorts, The Cancer Genome Atlas (TCGA) cohort, and a cohort of rectal cancers from the Beaumont RCSI Cancer Centre (BRCC). Multiplexed immunofluorescence staining of a tumour microarray (TMA) from the BRCC cohort was undertaken using Cell DIVE technology. Our cohorts included 87 cases (13.3%) of mucinous and 565 cases (86.7%) of non-mucinous CRC. Mucinous CRC in the TCGA dataset was associated with an increased proportion of CD8 + lymphocytes ( p = 0.018), regulatory T-cells ( p = 0.001) and M2 macrophages ( p = 0.001). In the BRCC cohort, mucinous RC was associated with enhanced CD8 + lymphocyte ( p = 0.022), regulatory T-cell ( p = 0.047), and B-cell ( p = 0.025) counts. High Fusobacterium abundance was associated with an increased proportion of CD4 + lymphocytes ( p = 0.031) and M1 macrophages ( p = 0.006), whilst M2 macrophages ( p = 0.043) were under-represented in this cohort. Patients with increased Fusobacterium relative abundance in our mucinous CRC TCGA cohort tended to have better clinical outcomes (DSS: likelihood ratio p = 0.04, logrank p = 0.052). Fusobacterium abundance may be associated with improved outcomes in mucinous CRC, possibly due to a modulatory effect on the host immune response. Key messages • Increased Fusobacterium relative abundance was not found to be associated with microsatellite instability in mucinous CRC. • Increased Fusobacterium relative abundance was associated with an M2/M1 macrophage switch, which is especially significant in mucinous CRC, where M2 macrophages are overexpressed. • Increased Fusobacterium relative abundance was associated with a significant improvement in disease specific survival in mucinous CRC. • Our findings were validated at a protein level within our own in house mucinous and non-mucinous rectal cancer cohorts.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

The Bcl-2 family proteins BAK and BAX control the crucial step of pore formation in the mitochondrial outer membrane during intrinsic apoptosis. Bcl-2-related ovarian killer (BOK) is a Bcl-2 family protein with a high sequence similarity to BAK and BAX. However, intrinsic apoptosis can proceed in the absence of BOK. Unlike BAK and BAX, BOK is primarily located on the endoplasmic reticulum (ER) and Golgi membranes, suggesting a role for BOK in regulating ER homeostasis. In this study, we report that BOK is required for a full ER stress response. Employing previously characterized fluorescent protein-based ER stress reporter cell systems, we show that BOK-deficient cells have an attenuated response to ER stress in all three signaling branches of the unfolded protein response. Fluo-4-based confocal Ca2+ imaging revealed that disruption of ER proteostasis in BOK-deficient cells was not linked to altered ER Ca2+ levels. Fluorescence recovery after photobleaching (FRAP) experiments using GRP78/BiP-eGFP demonstrated that GRP78 motility was significantly lower in BOK-deficient cells. This implied that less intraluminal GRP78 was freely available and more of the ER chaperone bound to unfolded proteins. Collectively, these experiments suggest a new role for BOK in the protection of ER proteostasis and cellular responses to ER stress.

Metabolic reprogramming is a hallmark of cancer. Somatic mutations in genes involved in oncogenic signaling pathways, including KRAS and TP53, rewire the metabolic machinery in cancer cells. We here set out to determine, at the single cell level, metabolic signatures in human colon cancer cells engineered to express combinations of activating KRAS gene mutations and TP53 gene deletions. Specifically, we explored how somatic mutations in these genes and substrate availability (lactate, glucose, substrate deprivation) from the extracellular microenvironment affect bioenergetic parameters, including cellular ATP, NADH and mitochondrial membrane potential dynamics. Employing cytosolic and mitochondrial FRET-based ATP probes, fluorescent NADH sensors, and the membrane-permeant cationic fluorescent probe TMRM in HCT-116 cells as a model system, we observed that TP53 deletion and KRAS mutations drive a shift in metabolic signatures enabling lactate to become an efficient metabolite to replenish both ATP and NADH following nutrient deprivation. Intriguingly, cytosolic, mitochondrial and overall cellular ATP measurements revealed that, in WT KRAS cells, TP53 deficiency leads to an enhanced ATP production in the presence of extracellular lactate and glucose, and to the greatest increase in ATP following a starvation period. On the other hand, oncogenic KRAS in TP53-deficient cells reversed the alterations in cellular ATP levels. Moreover, cell population measurements of mitochondrial and glycolytic metabolism using a Seahorse analyzer demonstrated that WT KRAS TP53-silenced cells display an increase of the basal respiration and tightly-coupled mitochondria, in the presence of glucose as substrate, compared to TP53 competent cells. Furthermore, cells possessing oncogenic KRAS, independently of TP53 status, showed less pronounced mitochondrial membrane potential changes in response to metabolic nutrients. Furthermore, analysis of cytosolic and mitochondrial NADH levels revealed that the simultaneous presence of TP53 deletion and oncogenic KRAS showed the most pronounced alteration in cytosolic and mitochondrial NADH during metabolic stress. In conclusion, our findings demonstrate how activating KRAS mutation and loss of TP53 remodel cancer metabolism and lead to alterations in bioenergetics under metabolic stress conditions by modulating cellular ATP production, NADH oxidation, mitochondrial respiration and function.

Entosis is a process where a living cell launches an invasion into another living cell's cytoplasm. These inner cells can survive inside outer cells for a long period of time, can undergo cell division, or can be released. However, the fate of most inner cells is lysosomal degradation by entotic cell death. Entosis can be detected by imaging a combination of membrane, cytoplasmic, nuclear, and lysosomal staining in the cells. Here, we provide a protocol for detecting entosis events and measuring the kinetics of entotic cell death by time-lapse imaging using tetramethylrhodamine methyl ester (TMRM) staining. This protocol was validated in: J Cell Biol (2021), DOI: 10.1083/jcb.202010030.

Abstract There is currently an urgent need to identify factors predictive of immunogenicity in colorectal cancer (CRC). Mucinous CRC is a distinct histological subtype of CRC, associated with a poor response to chemotherapy. Recent evidence suggests the commensal facultative anaerobe Fusobacterium may be especially prevalent in mucinous CRC. The objectives of this study were to assess the impact of Fusobacterium prevalence on immune cell expression and prognosis in mucinous CRC. Our study included two independent colorectal cancer patient cohorts, The Cancer Genome Atlas (TCGA) cohort, and a cohort of rectal cancers from the Beaumont RCSI Cancer Centre (BRCC). Multiplexed immunofluorescence staining of a tumor microarray (TMA) from the BRCC cohort was undertaken using Cell DIVE technology. Our cohorts included 87 cases (13.3%) of mucinous and 565 cases (86.7%) of non-mucinous CRC. Mucinous CRC in the TCGA dataset was associated with increased CD8 + lymphocyte (p = 0.018), regulatory T-cell (p = 0.001) and M2 macrophage (p = 0.001) expression. Similarly in the BRCC cohort, mucinous RC was associated with enhanced CD8 + lymphocyte (p = 0.022), regulatory T-cell (p = 0.047), and B-cell (p = 0.025) counts. Elevated Fusobacterium expression was associated with increased CD4+ (p = 0.031) and M1 macrophage (p = 0.006) expression, whilst M2 macrophages (p = 0.043) were under-expressed in the TCGA cohort. Increased Fusobacterium relative abundance in mucinous CRC was associated with improved clinical outcomes in our TCGA cohort despite having no association with MSI status (DSS: likelihood ratio p = 0.04, logrank p = 0.052). Fusobacterium abundance is associated with improved outcomes in mucinous CRC, possibly due its modulatory effect on the host immune response.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer without a targeted form of therapy. Unfortunately, up to 70% of patients with TNBC develop resistance to treatment. A known contributor to chemoresistance is dysfunctional mitochondrial apoptosis signaling. We set up a phenotypic small-molecule screen to reveal vulnerabilities in TNBC cells that were independent of mitochondrial apoptosis. Using a functional genetic approach, we identified that a “hit” compound, BAS-2, had a potentially similar mechanism of action to histone deacetylase inhibitors (HDAC). An in vitro HDAC inhibitor assay confirmed that the compound selectively inhibited HDAC6. Using state-of-the-art acetylome mass spectrometry, we identified glycolytic substrates of HDAC6 in TNBC cells. We confirmed that inhibition or knockout of HDAC6 reduced glycolytic metabolism both in vitro and in vivo. Through a series of unbiased screening approaches, we have identified a previously unidentified role for HDAC6 in regulating glycolytic metabolism.

Progressive neuronal injury following ischaemic stroke is associated with glutamate-induced depolarization, energetic stress and activation of AMP-activated protein kinase (AMPK). We here identify a molecular signature associated with neuronal AMPK activation, as a critical regulator of cellular response to energetic stress following ischaemia. We report a robust induction of microRNA miR-210-3p both in vitro in primary cortical neurons in response to acute AMPK activation and following ischaemic stroke in vivo. Bioinformatics and reverse phase protein array analysis of neuronal protein expression changes in vivo following administration of a miR-210-3p mimic revealed altered expression of phosphatase and tensin homolog (PTEN), 3-phosphoinositide-dependent protein kinase 1 (PDK1), ribosomal protein S6 kinase (p70S6K) and ribosomal protein S6 (RPS6) signalling in response to increasing miR-210-3p. In vivo, we observed a corresponding reduction in p70S6K activity following ischaemic stroke. Utilizing models of glutamate receptor over-activation in primary neurons, we demonstrated that induction of miR-210-3p was accompanied by sustained suppression of p70S6K activity and that this effect was reversed by miR-210-3p inhibition. Collectively, these results provide new molecular insight into the regulation of cell signalling during ischaemic injury, and suggest a novel mechanism whereby AMPK regulates miR-210-3p to control p70S6K activity in ischaemic stroke and excitotoxic injury.

Glioma stem cells (GSCs) are tumour initiating cells which contribute to treatment resistance, temozolomide (TMZ) chemotherapy and radiotherapy, in glioblastoma (GBM), the most aggressive adult brain tumour. A major contributor to the uncontrolled tumour cell proliferation in GBM is the hyper activation of cyclin-dependent kinases (CDKs). Due to resistance to standard of care, GBMs relapse in almost all patients. Targeting GSCs using transcriptional CDK inhibitors, CYC065 and THZ1 is a potential novel treatment to prevent relapse of the tumour. TCGA-GBM data analysis has shown that the GSC markers, CD133 and CD44 were significantly upregulated in GBM patient tumours compared to non-tumour tissue. CD133 and CD44 stem cell markers were also expressed in gliomaspheres derived from recurrent GBM tumours. Light Sheet Florescence Microscopy (LSFM) further revealed heterogeneous expression of these GSC markers in gliomaspheres. Gliomaspheres from recurrent tumours were highly sensitive to transcriptional CDK inhibitors, CYC065 and THZ1 and underwent apoptosis while being resistant to TMZ. Apoptotic cell death in GSC subpopulations and non-stem tumour cells resulted in sphere disruption. Collectively, our study highlights the potential of these novel CKIs to induce cell death in GSCs from recurrent tumours, warranting further clinical investigation.