The heterogeneity and the complex cellular architecture have a crucial effect on breast cancer progression and response to treatment. However, deciphering the neoplastic subtypes and their spatial organization is still challenging. Here, we combine single-nucleus RNA sequencing (snRNA-seq) with a microarray-based spatial transcriptomics (ST) to identify cell populations and their spatial distribution in breast cancer tissues. Malignant cells are clustered into distinct subpopulations. These cell clusters not only have diverse features, origins and functions, but also emerge to the crosstalk within subtypes. Furthermore, we find that these subclusters are mapped in distinct tissue regions, where discrepant enrichment of stromal cell types are observed. We also inferred the abundance of these tumorous subpopulations by deconvolution of large breast cancer RNA-seq cohorts, revealing differential association with patient survival and therapeutic response. Our study provides a novel insight for the cellular architecture of breast cancer and potential therapeutic strategies.

Background: APOBEC-driven mutagenesis and functional positive selection of mutated genes may synergistically drive the higher frequency of some hotspot driver mutations compared to other mutations within the same gene, as we reported forFGFR3S249C. Only a few APOBEC-associated driver hotspot mutations have been identified in bladder cancer (BCa). Here, we systematically looked for and characterised APOBEC-associated hotspots in BCa.Methods: We analysed 602 published exome-sequenced BCas, for part of which gene expression data were also available. APOBEC-associated hotspots were identified by motif-mapping, mutation signature fitting and APOBEC-mediated mutagenesis comparison. Joint analysis of DNA hairpin stability and gene expression was performed to predict driver or passenger hotspots. Aryl hydrocarbon receptor (AhR) activity was calculated based on its target genes expression. Effects of AhR knockout/inhibition on BCa cell viability were analysed.Results: We established a panel of 44 APOBEC-associated hotspot mutations in BCa, which accounted for about half of the hotspot mutations. Fourteen of them overlapped with the hotspots found in other cancer types with high APOBEC activity. They mostly occurred in the DNA lagging-strand templates and the loop of DNA hairpins. APOBEC-associated hotspots presented systematically a higher prevalence than the other mutations within each APOBEC-target gene, independently of their functional impact. A combined analysis of DNA loop stability and gene expression allowed to distinguish known passenger from known driver hotspot mutations in BCa, including loss-of-function mutations affecting tumour suppressor genes, and to predict new candidate drivers, such asAHRQ383H. We further characterisedAHRQ383H as an activating driver mutation associated with high AhR activity in luminal tumours. High AhR activity was also found in tumours presenting amplifications ofAHRand its co-receptorARNT. We finally showed that BCa cells presenting those different genetic alterations were sensitive to AhR inhibition.Conclusions: Our study identified novel potential drivers within APOBEC-associated hotspot mutations in BCa reinforcing the importance of APOBEC mutagenesis in BCa. It could allow a better understanding of BCa biology and aetiology and have clinical implications such as AhR as a potential therapeutic target. Our results also challenge the dogma that all hotspot mutations are drivers and mostly gain-of-function mutations affecting oncogenes.

Background: Although immunotherapy is effective in improving the clinical outcomes of patients with bladder cancer (BC), it is only effective in a small percentage of patients. Intercellular crosstalk in the tumor microenvironment strongly influences patient response to immunotherapy, while the crosstalk patterns of plasma cells (PCs) as endogenous antibody-producing cells remain unknown. Here, we aimed to explore the heterogeneity of PCs and their potential crosstalk patterns with BC tumor cells. Methods: Crosstalk patterns between PCs and tumor cells were revealed by performing integrated bulk and single-cell RNA sequencing (RNA-seq) and spatial transcriptome data analysis. A risk model was constructed based on ligand/receptor to quantify crosstalk patterns by stepwise regression Cox analysis. Results: Based on cell infiltration scores inferred from bulk RNA-seq data (n = 728), we found that high infiltration of PCs was associated with better overall survival (OS) and response to immunotherapy in BC. Further single-cell transcriptome analysis (n = 8; 41,894 filtered cells) identified two dominant types of PCs, IgG1 and IgA1 PCs. Signal transduction from tumor cells of specific states (stress-like and hypoxia-like tumor cells) to PCs, for example, via the LAMB3/CD44 and ANGPTL4/SDC1 ligand/receptor pairs, was validated by spatial transcriptome analysis and associated with poorer OS as well as nonresponse to immunotherapy. More importantly, a ligand/receptor pair-based risk model was constructed and showed excellent performance in predicting patient survival and immunotherapy response. Conclusions: PCs are an important component of the tumor microenvironment, and their crosstalk with tumor cells influences clinical outcomes and response to immunotherapies in BC patients. Graphical

Background: Bladder cancer is a common malignancy that affects the human urinary tract. Muscle-invasive bladder cancer (MIBC) is aggressive and has poor prognosis. Previous studies have reported that the tumor-infiltrating lymphocytes (TILs) were associated with MIBC outcome; however, inconsistency remains and mRNA level TIL markers’ prognostic significance in MIBC is unclear. Materials and methods: In the present study, we reanalyzed data from four public datasets (the Cancer Genome Atlas for investigation; and CIT, GSE5287, and GSE31684 for validation) to examine the prognostic significance of CD3D, CD4, CD8A, CD3D/CD4 and CD3D/CD8A in MIBC. Results: We found that the CD3D/CD4 ratio was a stable independent prognostic factor in MIBC (beta = −0.87, P = 0.025); high CD3D/CD4 ratio predicted better survival in MIBC, and the power of this association was much stronger in basal-squamous tumors (beta = −4.73, P = 2.67E-06). We also noted that the CD4 expression was significantly higher than CD3D (P < 0.05), indicating the presence of CD3^−,CD4^+, cells which could be immune-suppressing. Conclusion: The CD3D/CD4 ratio can be viewed as a prognostic marker and a rough measurement for the interaction between immune-effecting CD3^+, TILs and immune-suppressing CD3^−,CD4^+, cells in MIBC, and this interaction may play a particularly important role in anti-cancer immunity in basal-squamous tumors as it has a very strong association with survival in this subtype, and may be used to select potential responders to immunotherapy.

Background: The stem or progenitor antecedents confer developmental plasticity and unique cell identities to cancer cells via genetic and epigenetic programs. A comprehensive characterization and mapping of the cell-of-origin of breast cancer using novel technologies to unveil novel subtype-specific therapeutic targets is still absent.

Liquid biopsies, in particular, analysis of cell-free DNA, are expected to revolutionize the current landscape of cancer diagnostics and treatment. However, the existing methods for cfDNA-based liquid biopsies for cancer have certain limitations, such as fragment interruption and GC bias, which are likely to be resolved by the emerging Oxford Nanopore Technologies (ONT), characterized by long read-length, fast read-times, high throughput, and polymerase chain reaction-free. In this review, we summarized the current literatures regarding the feasibility and applications of cfDNA-based liquid biopsies using ONT for cancer management, a possible game-changer that we believe is promising in detecting multimodal biomarkers and can be applied in a wide range of oncology utilities including early screening, diagnosis, and treatment monitoring.