Ferroptosis is a form of programmed cell death that is modulated in some cancer cells as a pro-survival mechanism. Induction of ferroptosis is a potential anti-cancer strategy, and enhancement of ferroptosis using ferroptosis inducers has the potential to enhance current anti-tumour mechanisms. In this study, we assessed the effect of the ferroptosis inducers Erastin, RSL-3 and FIN-56 on radiosensitivity in 2D cell culture, and in 3D alginate tumour spheroids from breast cancer cell lines. Since some tumours modulate ferroptosis via increased Nrf2 production, and MCF-7 and MDA-MB-231 both produce Nrf2 protein, we also assessed the effects of the Nrf2 inhibitor ML385 on radiosensitivity. MDA-MB-231 was highly sensitive to all ferroptosis inducers, and ferroptosis was reversed by the ferroptosis inhibitors Ferrostatin-1, Liproxstatin-1 and Deferoxamine. MCF-7 was resistant to all ferroptosis inducers. MDA-MB-231 and MCF-7 cells were sensitive to irradiation in 2D cell culture but resistant to irradiation in 3D alginate spheroids. Ferroptosis inducers did not synergistically enhance irradiation-induced cell death in 2D cell cultures. There was also no robust enhancement to irradiation effects with ferroptosis inducers in 2D or 3D cell culture. Ferroptosis inducers did, however, show a heterogeneous response in 3D cell culture, in that isogenic spheroids responded differently within the same spheroid. The Nrf2 inhibitor ML385 showed no synergistic enhancement of ferroptotic cell death when combined with irradiation. These studies suggest targeting ferroptosis does not induce short-term enhancement of ferroptotic cell death. less

Emerging imaging spatial transcriptomics (iST) platforms and coupled analytical methods can recover cell-to-cell interactions, groups of spatially covarying genes, and gene signatures associated with pathological features, and are thus particularly well-suited for applications in formalin fixed paraffin embedded (FFPE) tissues. Here, we benchmarked the performance of three commercial iST platforms on serial sections from tissue microarrays (TMAs) containing 23 tumor and normal tissue types for both relative technical and biological performance. On matched genes, we found that 10X Xenium shows higher transcript counts per gene without sacrificing specificity, but that all three platforms concord to orthogonal RNA-seq datasets and can perform spatially resolved cell typing, albeit with different false discovery rates, cell segmentation error frequencies, and with varying degrees of sub-clustering for downstream biological analyses. Taken together, our analyses provide a comprehensive benchmark to guide the choice of iST method as researchers design studies with precious samples in this rapidly evolving field. less

Background Acute myeloid leukaemia (AML) is a deadly haematological malignancy that originates from mutated myeloid progenitor cells that lie quiescent in the hypoxic bone marrow. Elderly patients who cannot tolerate standard chemotherapies are administered low-dose hypomethylating agents (HMA) which act in a replication-dependent manner to reprogram the epigenome. Relapse is common following HMA treatment and may arise from quiescent leukaemia cells in the hypoxic bone marrow. Therefore, the effects of hypoxia on HMA efficacy may influence AML progression. Results AML cell lines (MOLM-13, MV-4-11, HL-60) were treated with decitabine (100nM) or azacitidine (500-2000nM) in normoxic (21% O2) and hypoxic (1% O2) conditions. Exposure to hypoxia significantly reduced AML cell growth across all cell lines, with no additional effects observed upon HMA treatment. This was associated with distinct effects on DNA methylation. The extent of hypomethylation induced by AZA treatment was reduced in hypoxia, whereas DAC-induced hypomethylation was maintained in low oxygen conditions. Transcriptional response to HMA treatment were also altered in hypoxia, with HMAs failing to up-regulate antigen presentation pathways in hypoxia. In particular, human leukocyte antigens (HLAs) such as HLA-DR were increased upon HMA treatment in normoxia, but not hypoxia. Conclusion Our results suggest that HMA-induced antigen presentation may be impaired in hypoxic tissues such as the bone marrow. This study highlights the need to consider microenvironmental factors when designing co-treatment strategies to improve HMA therapeutic efficacy. less

Colorectal cancer (CRC) patients have been shown to possess an altered gut microbiome. Diet is a known microbiome modulator. An attenuating effect of the ketogenic diet (KD) on CRC cell growth has been previously observed, however the role of the gut microbiome in driving this effect remains unknown. Here, we describe a reduced colonic tumor burden upon KD consumption in a CRC mouse model with a humanized microbiome. Importantly, we demonstrate a causal relationship through microbiome transplantation into germ-free mice, whereby alterations in gut microbial function were maintained in the absence of continued selective pressure from the KD. Stearic acid is identified as a putative microbiome derived anti-cancer metabolite. Taken together, the beneficial effects of the KD are mediated by the gut microbiome and may be dependent on a pre-existing non-responder or responder gut microbiome profile. These results have important implications for future clinical trials evaluating the effects of the ketogenic diet on human CRC. less

Clonal lineage inference (\"tumor phylogenetics\") has become a crucial tool for making sense of somatic evolution processes that underlie cancer development and are increasingly recognized as part of normal tissue growth and aging. The inference of clonal lineage trees from single cell sequence data offers particular promise for revealing processes of somatic evolution in unprecedented detail. However, most such tools are based on fairly restrictive models of the types of mutation events observed in somatic evolution and of the processes by which they develop. The present work seeks to enhance the power and versatility of tools for single-cell lineage reconstruction by making more comprehensive use of the range of molecular variant types by which tumors evolve. We introduce Sc-TUSV-ext, an integer linear programming (ILP) based tumor phylogeny reconstruction method that, for the first time, integrates single nucleotide variants (SNV), copy number alterations (CNA) and structural variations (SV) into clonal lineage reconstruction from single-cell DNA sequencing data. We show on synthetic data that accounting for these variant types collectively leads to improved accuracy in clonal lineage reconstruction relative to prior methods that consider only subsets of the variant types. We further demonstrate the effectiveness on real data in resolving clonal evolution in the presence of multiple variant types, providing a path towards more comprehensive insight into how various forms of somatic mutability collectively shape tissue development. less

Osteosarcoma is the most prevalent pediatric solid bone tumor. These tumors are highly metastatic and frequently develop resistance to chemotherapy, leading to poor survival rate for patients. We found that C-terminal Binding Protein 2 (CtBP2) and Cysteine-rich protein 61 (CYR61/CCN1) expression level correlated positively in a panel of cell lines. In silico analysis of protein-protein interaction network revealed a link with stemness markers. We confirmed that CtBP2 influences stemness markers expression, cell clonogenicity, cell migration, matrix metalloproteinase activity and cell invasion. Surprisingly, using syngeneic tumor cells graft models, while induction of CtBP2 expression correlated with the metastatic dissemination process, it occurred only at the invasive front. Hypoxic conditions in central tumor region interfered with CtBP2 induction. Globally, we identify for the first time that CtBP2 is a required inducing factor in the CYR61-related metastatic progression of osteosarcoma. Moreover, we demonstrate that while CtBP2 is a downstream transcriptional target of CYR61 signaling cascade, it occurs only under non-hypoxic conditions. The present study suggests that CtBP2 may represent a potential pivotal target for therapeutic management of metastases spreading in osteosarcoma. less

Uveal melanoma (UM) is an ocular tumor that often develops asymptomatically. Statistically, every second patient eventually develops metastases that drastically worsen the prognosis by several months of overall survival. While isolated liver perfusion with melphalan and more recently immunotherapy (Tebentafusp) are the few treatment options available for metastatic UM patients, their application is complex or expensive. There is an urgent need to understand drug response and identify potential avenues for therapy. Hence, we focused on uncovering altered phosphorylation signaling events in metastatic UM using proteomics as an approach to identify potential drug targets. We analyzed the phosphoproteomes of the primary UM cell line Mel270 and two cell lines OMM2.3 and OMM2.5, derived from metastatic lesions of the same patient. We found 177 phosphosites to be altered significantly between primary and metastatic cell lines. Pathway analysis of up-regulated phosphosites in metastatic lines suggests that Rho signaling and mitotic cell cycle to be significantly altered uncovering potential routes of signaling for metastasis. Clinical data from LUMC and TCGA datasets uncovered MARK3 expression (which links to Rho signaling) correlation with chromosome 3 status, a prognostic marker in UM, suggesting that MARK3 kinase might be involved in metastatic UM signaling. less

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide and further research on its biology is needed to fully understand the disease. Dysregulation of alternative RNA splicing is a common hallmark in cancer, including PDAC, which provides an emerging source of knowledge and of novel biomarkers and therapeutic tools. Here, we examined the role of EIF4A3, a core component of the Exon Junction Complex intimately linked to RNA splicing, in pancreatic cancer biology. EIF4A3 is overexpressed in PDAC tissue and associated to clinical parameters of malignancy and poorer patient survival. Mechanistically, exploration of PDAC RNA-seq data unveiled the link of EIF4A3 to diverse malignancy processes, in line with its association to key molecular pathways. Accordingly, EIF4A3 targeting in vitro decreased essential functional tumor features such as proliferation, migration, colony formation and sphere formation, while its in vivo targeting reduced tumor growth. EIF4A3 silencing in PDAC cell lines severely altered its transcriptional and spliceosomic landscapes, as shown by RNA-seq analyses, suggesting a role for EIF4A3 in maintaining RNA homeostasis. Our results indicate that EIF4A3 dysregulation in PDAC has a pleiotropic regulatory role on RNA biology, influencing key cellular functions. This paves the way to explore its potential as a novel biomarker and actionable target candidate for this lethal cancer. less

Breast cancer exhibits significant heterogeneity, manifesting in various subtypes that are critical in guiding treatment decisions. This study aimed to investigate the existence of distinct subtypes of breast cancer within the Asian population, by analysing the transcriptomic profiles of 934 breast cancer patients from a Malaysian cohort. Our findings reveal that the HR+/HER2- breast cancer samples display a distinct clustering pattern based on immune phenotypes, rather than conforming to the conventional luminal A-luminal B paradigm previously reported in breast cancers from women of European descent. This suggests that the activation of the immune system may play a more important role in Asian HR+/HER2- breast cancer than has been previously recognized. Analysis of somatic mutations by whole exome sequencing showed that counter-intuitively, the cluster of HR+/HER2- samples exhibiting higher immune scores was associated with lower tumour mutational burden, lower homologous recombination deficiency scores, and fewer copy number aberrations, implicating the involvement of non-canonical tumour immune pathways. Further investigations are warranted to determine the underlying mechanisms of these pathways, with the potential to develop innovative immunotherapeutic approaches tailored to this specific patient population. less

Deregulated nucleotide metabolism, and in particular increased pyrimidine metabolism (PyMet), has been shown to contribute to various pathological features of cancer including chemoresistance and epithelial-to-mesenchymal transition. However, cancer often encompasses complex signaling and metabolic pathway cascades for its progression, and understanding of these molecular regulatory processes in pyrimidine metabolism is quite limited. Therefore, a comprehensive pan-cancer analysis in around 10,000 gene expression profiles of 32 cancer types was employed using a pathway-based approach utilizing gene-sets representing various signaling and metabolic pathways. The analysis identified several top connections with PyMet including TERT, MTOR, DAX1, HOXA1, TP53 and TNC implying an inter-dependency of regulations which in turn was linked to the chemoresistance mechanisms. PyMet-signaling interactions were validated with in vitro derived gene-sets from endogenous thymidylate synthase (TYMS)-promoter activity reporter, from TYMS knockdown and from brequinar treatment, and further at single cell transcriptome level. Strikingly, brequinar treatment profile showed a strong inverse association pattern with doxorubicin chemoresistance in multiple cancer types. The study highlights the PyMet-pathway interactions and its role in chemoresistance, thereby providing an effective tool for improving PyMet targeting strategy in cancer. The analysis as an accessible resource is available at: www.pype.compbio.sdu.dk less