While cancer immunotherapies have primarily focused on activation of cytotoxic CD8 cells, CD4 T cell activity is also associated with survival and immunotherapeutic response in numerous cancers. We applied integrated single-cell RNA sequencing and multiplexed protein epitope profiling to breast cancer samples to resolve the complexity of immune cell states within the tumor microenvironment. This approach enhanced phenotypic resolution, identifying three distinct states within the CD4 T follicular helper-like (Tfh) cell cluster. A CXCR4high progenitor state gave rise to two differentiated states: an IGFL2high subset resembling conventional Tfh cells and localised to B cell-rich lymphoid aggregates, and a CD103+ subset, exhibiting features of tissue residency, exhaustion, and cytotoxicity, which co-localised with tumor foci. CD103+ Tfh-like cells were found to interact with CXCL10+ macrophages through production of CCL chemokines and CSF1. A higher CD103+ Tfh to IGFL2high Tfh ratio, together with the selective clonal expansion of the CD103+ subset, was strongly associated with improved tumour immunity and superior responses to anti-PD-1 checkpoint blockade, surpassing the predictive value of exhausted CD8 T cells. These findings integrate Tfh and CD4 with cytotoxic potential in breast cancer, offering new insight into anti-tumor immunity and response to checkpoint blockade.

Abstract While cancer immunotherapies have primarily focused on activation of cytotoxic CD8 killing, CD4 T cell activity is also associated with survival and immunotherapeutic response in numerous cancers. We applied integrated single-cell RNA sequencing and multiplexed protein epitope profiling to breast cancer samples to resolve the complexity of immune cell states within the tumor microenvironment. This approach enhanced phenotypic resolution, identifying three distinct states within the T follicular helper (Tfh) cell cluster. A CXCR4high progenitor state gave rise to two differentiated states: an IGFL2high subset resembling conventional Tfh cells and localised to B cell-rich lymphoid aggregates, and a CD103+ subset, exhibiting features of tissue residency, exhaustion, and cytotoxicity, which co-localised with tumor foci. CD103+ Tfh-like cells were found to interact with CXCL10+ macrophages through production of CCL chemokines and CSF1. A higher CD103+ Tfh to IGFL2high Tfh ratio correlated with improved patient survival and enhanced responses to anti-PD1 checkpoint blockade. These findings integrate Tfh and CD4 with cytotoxic potential in breast cancer, offering new insight into anti-tumor immunity and response to checkpoint blockade. Citation Format: Ghamdan Al-Eryani, Sophie van der Leij, Etienne Masle-Farquhar, Alma Andersson, Kate Harvey, Sunny Wu, Tony Wang, John Reeves, Cindy Ma, Daniel L. Roden, Charles M. Perou, Nir Hacohen, Aziz Al’Khafaji, Mats Nilsson, Joakim Lundeberg, Marcel Batten, Simon Junankar, Alexander Swarbrick. Proteotranscriptomic dissection of breast cancer T cell states identifies CD103+ Tfh-derived cytotoxic CD4+ cells linked to immunotherapy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7411.

Lung cancers and melanomas have many somatically mutated self-proteins that would be expected to trigger an immune rejection response, yet therapeutic responses can only be induced in a subset of patients. Here, we investigated the possibility that inherited differences in immune tolerance checkpoints contribute to variability in outcomes. Whole genome sequencing revealed biallelic germline loss-of-function (LOF) mutations in the immune tolerance checkpoint gene, NOD2, in an exceptional immune responder to targeted radiotherapy for metastatic melanoma. In 40 exceptional immune responders to anti-PD1 monotherapy for non-small cell lung cancer (NSCLC), genome sequencing showed 30% had inherited a NOD2 LOF variant, more than twice the population frequency (P = 0.0021). Conversely, a gain-of-function RIPK2 allele known to increase NOD2 signaling was inherited by 61% of nonresponders from the same cohort, compared to 10% of exceptional responders and much higher than the population frequency (P < 0.0001). Within the overall recruited cohort of 144 NSCLC anti-PD1 patients, individuals with immune-related adverse events (irAE) had better overall survival, further improved in those with NOD2 LOF. In independent anti-PD1 monotherapy cohorts with a range of cancers, inherited NOD2 LOF was associated with complete or partial response (P = 0.0107). Experimental validation in mice showed germline Nod2 LOF enhanced therapeutic immune responses elicited by anti-PD1 monotherapy against a high mutation burden colorectal cancer, increasing tumor infiltration by effector memory CD8 T cells. Collectively these results reveal common inherited human variation in an immune tolerance checkpoint is a determinant of cancer immune responses elicited by pharmacological inhibition of another checkpoint.

In recent decades, the role of tumor biomechanics on cancer cell behavior at the primary site has been increasingly appreciated. However, the effect of primary tumor biomechanics on the latter stages of the metastatic cascade, such as metastatic seeding of secondary sites and outgrowth remains underappreciated. This work sought to address this in the context of triple negative breast cancer (TNBC), a cancer type known to aggressively disseminate at all stages of disease progression. Using mechanically tuneable model systems, mimicking the range of stiffness's typically found within breast tumors, it is found that, contrary to expectations, cancer cells exposed to softer microenvironments are more able to colonize secondary tissues. It is shown that heightened cell survival is driven by enhanced metabolism of fatty acids within TNBC cells exposed to softer microenvironments. It is demonstrated that uncoupling cellular mechanosensing through integrin β1 blocking antibody effectively causes stiff primed TNBC cells to behave like their soft counterparts, both in vitro and in vivo. This work is the first to show that softer tumor microenvironments may be contributing to changes in disease outcome by imprinting on TNBC cells a greater metabolic flexibility and conferring discrete cell survival advantages.

Real-time intravital two-photon microscopy showing phagocytosis of a large, granular clump of bisphosphonate by a single tumour-associated macrophage.

Real-time intravital two-photon microscopy showing pinocytosis of bisphosphonate by a single tumour-associated macrophage.

Real-time intravital two-photon microscopy showing distribution of bisphosphonate in normal breast tissue.

Real-time intravital two-photon microscopy showing distribution of bisphosphonate in 4T1 breast tumour.

3D reconstruction of 4T1 mammary tumour 24 hours after injection of bisphosphonate.

Cancers evade the immune system through the process of cancer immunoediting. While immune checkpoint inhibitors are effective for reactivating tumour immunity in some cancer types, many other solid cancers, including breast cancer, remain largely non-responsive. Understanding how non-responsive cancers evade immunity and whether this occurs at the clonal level will improve immunotherapeutic design. Here we use DNA barcoding to track murine mammary cancer cell clones during immunoediting and determine clonal transcriptional profiles that allow immune evasion following anti-PD1 plus anti-CTLA4 immunotherapy. Clonal diversity is significantly restricted by immunotherapy treatment in both primary tumours and metastases, demonstrating selection for pre-existing breast cancer cell populations and ongoing immunoediting during metastasis and treatment. Immunotherapy resistant clones express a common gene signature associated with poor survival of basal-like breast cancer patient cohorts. At least one of these genes has an existing small molecule that can potentially be used to improve immunotherapy response.

Summary Immune cells are critical determinants of solid tumour aetiology, but the diverse phenotypes of intra-tumoural immune cells remain incompletely characterised. We applied integrated single cell RNA sequencing (scRNA-Seq) and highly multiplexed protein epitope analysis to a cohort of breast cancer samples to resolve cell states within the tumour microenvironment. We reveal novel protein markers for resting and activated tumour infiltrating lymphocytes, and show that high expression of CD103 primarily marks exhausted CD8 rather than tissue resident CD8 T-cells in human breast cancers. We identify two distinct states of activated CD4+ T follicular helper (Tfh) cells. A population resembling conventional Tfh (cTfh) cells were localised primarily to lymphoid aggregates by spatial transcriptomics. In contrast, cancer associated Tfh (caTfh) cells expressing markers of tissue residency and exhaustion co-localized with cancer foci and signalled to macrophages. Importantly, increased caTfh : cTfh ratio associated with improved disease outcome and response to checkpoint immunotherapy.

Inhibitor of differentiation (ID) proteins dimerize with basic HLH (bHLH) transcription factors, repressing transcription of lineage-specification genes across diverse cellular lineages. ID4 is a key regulator of mammary stem cells; however, the mechanism by which it achieves this is unclear. Here, we show that ID4 has a cell autonomous role in preventing myoepithelial differentiation of basal cells in mammary organoids and in vivo. ID4 positively regulates proliferative genes and negatively regulates genes involved in myoepithelial function. Mass spectrometry reveals that ID4 interacts with the bHLH protein HEB, which binds to E-box motifs in regulatory elements of basal developmental genes involved in extracellular matrix and the contractile cytoskeleton. We conclude that high ID4 expression in mammary basal stem cells antagonizes HEB transcriptional activity, preventing myoepithelial differentiation and allowing for appropriate tissue morphogenesis. Downregulation of ID4 during pregnancy modulates gene regulated by HEB, promoting specialization of basal cells into myoepithelial cells.

Breast cancers are complex cellular ecosystems where heterotypic interactions play central roles in disease progression and response to therapy. However, our knowledge of their cellular composition and organization is limited. Here we present a single-cell and spatially resolved transcriptomics analysis of human breast cancers. We developed a single-cell method of intrinsic subtype classification (SCSubtype) to reveal recurrent neoplastic cell heterogeneity. Immunophenotyping using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) provides high-resolution immune profiles, including new PD-L1/PD-L2+ macrophage populations associated with clinical outcome. Mesenchymal cells displayed diverse functions and cell-surface protein expression through differentiation within three major lineages. Stromal-immune niches were spatially organized in tumors, offering insights into antitumor immune regulation. Using single-cell signatures, we deconvoluted large breast cancer cohorts to stratify them into nine clusters, termed 'ecotypes', with unique cellular compositions and clinical outcomes. This study provides a comprehensive transcriptional atlas of the cellular architecture of breast cancer.

Abstract Breast cancers are complex cellular ecosystems where heterotypic interactions play central roles in disease progression and response to therapy. However, our knowledge of the cellular composition and organization of breast cancer remains limited. We present a comprehensive single cell and spatially resolved transcriptomic atlas of human breast cancers. The 10X Genomics Chromium platform was used to generate single cell transcriptomic data (scRNA-Seq) from more than 120,000 cells sampled from 26 breast cancers. CITE-Seq was employed to simultaneously generate protein measurements using a panel of 157 antibodies against immune, stromal and epithelial cell surface markers and analysed using Seurat. Using single cell signatures, we estimated the cellular composition of more than 2000 breast cancers in the Metabric cohort using deconvolution methods. Spatial transcriptomics was conducted on 12 frozen tissues (Luminal, Her2+ and triple negative breast cancer (TNBC)) using the 10X genomics Visium solution. We also used a novel Spatial Whole Transcriptome Panel, targeting 18,000+ genes on the Nanostring GeoMX platform, to profile T cells and malignant cells across multiple tissue niches from 16 TNBC FFPE cases. Integrative scRNA-Seq analysis identifies recurrent gene modules driving neoplastic cell heterogeneity, including interferon signaling, estrogen receptor function and mutually exclusive patterns of proliferation versus EMT. We also develop a single cell classifier of intrinsic subtype (scSubtype) to reveal frequent intra-tumoral heterogeneity for breast cancer intrinsic subtypes. CITE-Seq revealed immune profiles at high resolution, leading to the identification of novel macrophage populations with high expression of PD-L1 and PD-L2 immune checkpoint ligands and associations with clinical outcome. We also observe enrichment of exhausted and proliferative CD8 T cells in TNBC, with unique patterns of cell-surface checkpoint protein expression when compared to other subtypes. Targeted analysis using the GeoMX revealed spatial segregation of T cell phenotypes, with exhausted and proliferative CD8 T cells forming small clusters adjacent to tumor cells with high interferon pathway activity. Analysis of scRNA-Seq data revealed that stromal cells generate diverse functions and cell surface protein expression through differentiation within 3 major lineages: fibroblast, endothelial and perivascular-like. Subsets of stromal cells had features associated with immune regulation and Visium data revealed that stromal-immune niches were spatially organized in tumors, offering insights into anti-tumor immune suppression by stromal cells. Finally, deconvolution stratified >2000 breast cancer cases in Metabric into nine clusters, termed ‘ecotypes', with distinct cellular compositions and clinical outcomes. This study provides a comprehensive atlas of the cellular architecture of breast cancer. Citation Format: Sunny Z. Wu, Daniel Roden, Ghamdan Al Eryani, Simon Junankar, Elgene Lim, Aatish Thennavan, Alma Andersson, Stephen Williams, Jingjing Gong, Robin Fropf, Kit Fuhrman, Joakim Lundeberg, Chuck Perou, Alexander Swarbrick. An integrated multi-omic cellular atlas of human breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 129.

The tumour stroma regulates nearly all stages of carcinogenesis. Stromal heterogeneity in human triple-negative breast cancers (TNBCs) remains poorly understood, limiting the development of stromal-targeted therapies. Single-cell RNA sequencing of five TNBCs revealed two cancer-associated fibroblast (CAF) and two perivascular-like (PVL) subpopulations. CAFs clustered into two states: the first with features of myofibroblasts and the second characterised by high expression of growth factors and immunomodulatory molecules. PVL cells clustered into two states consistent with a differentiated and immature phenotype. We showed that these stromal states have distinct morphologies, spatial relationships and functional properties in regulating the extracellular matrix. Using cell signalling predictions, we provide evidence that stromal-immune crosstalk actsviaa diverse array of immunoregulatory molecules. Importantly, the investigation of gene signatures from inflammatory-CAFs and differentiated-PVL cells in independent TNBC patient cohorts revealed strong associations with cytotoxic T-cell dysfunction and exclusion, respectively. Such insights present promising candidates to further investigate for new therapeutic strategies in the treatment of TNBCs.

Background Basal-like breast cancer (BLBC) is a poorly characterised, heterogeneous disease. Patients are diagnosed with aggressive, high-grade tumours and often relapse with chemotherapy resistance. Detailed understanding of the molecular underpinnings of this disease is essential to the development of personalised therapeutic strategies. Inhibitor of differentiation 4 (ID4) is a helix-loop-helix transcriptional regulator required for mammary gland development. ID4 is overexpressed in a subset of BLBC patients, associating with a stem-like poor prognosis phenotype, and is necessary for the growth of cell line models of BLBC through unknown mechanisms. Methods Here, we have defined unique molecular insights into the function of ID4 in BLBC and the related disease high-grade serous ovarian cancer (HGSOC), by combining RIME proteomic analysis, ChIP-seq mapping of genomic binding sites and RNA-seq. Results These studies reveal novel interactions with DNA damage response proteins, in particular, mediator of DNA damage checkpoint protein 1 (MDC1). Through MDC1, ID4 interacts with other DNA repair proteins (γH2AX and BRCA1) at fragile chromatin sites. ID4 does not affect transcription at these sites, instead binding to chromatin following DNA damage. Analysis of clinical samples demonstrates that ID4 is amplified and overexpressed at a higher frequency in BRCA1 -mutant BLBC compared with sporadic BLBC, providing genetic evidence for an interaction between ID4 and DNA damage repair deficiency. Conclusions These data link the interactions of ID4 with MDC1 to DNA damage repair in the aetiology of BLBC and HGSOC.

The tumour stroma regulates nearly all stages of carcinogenesis. Stromal heterogeneity in human triple‐negative breast cancers (TNBCs) remains poorly understood, limiting the development of stromal‐targeted therapies. Single‐cell RNA sequencing of five TNBCs revealed two cancer‐associated fibroblast (CAF) and two perivascular‐like (PVL) subpopulations. CAFs clustered into two states: the first with features of myofibroblasts and the second characterised by high expression of growth factors and immunomodulatory molecules. PVL cells clustered into two states consistent with a differentiated and immature phenotype. We showed that these stromal states have distinct morphologies, spatial relationships and functional properties in regulating the extracellular matrix. Using cell signalling predictions, we provide evidence that stromal‐immune crosstalk acts via a diverse array of immunoregulatory molecules. Importantly, the investigation of gene signatures from inflammatory‐CAFs and differentiated‐PVL cells in independent TNBC patient cohorts revealed strong associations with cytotoxic T‐cell dysfunction and exclusion, respectively. Such insights present promising candidates to further investigate for new therapeutic strategies in the treatment of TNBCs. This single‐cell gene expression resource deciphers the composition of triple‐negative breast cancer (TNBC) stroma, revealing distinct subclasses of cancer‐associated fibroblasts (CAFs) and perivascular‐like (PVL) cells. These signatures are informative on tumour aetiology and potential strategies for development of targeted therapies. Single‐cell profiling of primary breast cancer provides unprecedented insights into cell‐type heterogeneity within the tumor microenvironment.

Breast cancers display phenotypic and functional heterogeneity and several lines of evidence support the existence of cancer stem cells (CSCs) in certain breast cancers, a minor population of cells capable of tumor initiation and metastatic dissemination. Identifying factors that regulate the CSC phenotype is therefore important for developing strategies to treat metastatic disease. The Inhibitor of Differentiation Protein 1 (Id1) and its closely related family member Inhibitor of Differentiation 3 (Id3) (collectively termed Id) are expressed by a diversity of stem cells and are required for metastatic dissemination in experimental models of breast cancer. In this study, we show that ID1 is expressed in rare neoplastic cells within ER-negative breast cancers. To address the function of Id1 expressing cells within tumors, we developed independent murine models of Triple Negative Breast Cancer (TNBC) in which a genetic reporter permitted the prospective isolation of Id1+ cells. Id1+ cells are enriched for self-renewal in tumorsphere assays in vitro and for tumor initiation in vivo. Conversely, depletion of Id1 and Id3 in the 4T1 murine model of TNBC demonstrates that Id1/3 are required for cell proliferation and self-renewal in vitro, as well as primary tumor growth and metastatic colonization of the lung in vivo. Using combined bioinformatic analysis, we have defined a novel mechanism of Id protein function via negative regulation of the Roundabout Axon Guidance Receptor Homolog 1 (Robo1) leading to activation of a Myc transcriptional programme.

Differentiation of stem cells embedded within the mammary epithelium is orchestrated by lineage-specifying transcription factors. Unlike the well-defined luminal hierarchy, dissection of the basal lineage has been hindered by a lack of specific markers. Inhibitor of Differentiation 4 (ID4) is a basally-restricted helix-loop-helix (HLH) transcription factor essential for mammary development. Here we show that ID4 is highly expressed in basal stem cells and decreases during myoepithelial differentiation. By integrating transcriptomic, proteomic, and ChIP-sequencing data, we reveal that ID4 is required to suppress myoepithelial gene expression and cell fate. We identify the bHLH protein HEB as a direct binding partner of ID4, and describe a previously-unknown role for this regulator in mammary development. HEB binds to E-boxes in regulatory elements of developmental genes, negatively regulated by ID4, involved in extracellular matrix synthesis and cytoskeletal contraction. Together our findings support a model whereby ID4 binds to HEB and blocks it from promoting myoepithelial specialisation. These new insights expand our current understanding into control of myoepithelial differentiation and mammary gland morphogenesis.

High-throughput single-cell RNA sequencing is a powerful technique but only generates short reads from one end of a cDNA template, limiting the reconstruction of highly diverse sequences such as antigen receptors. To overcome this limitation, we combined targeted capture and long-read sequencing of T-cell-receptor (TCR) and B-cell-receptor (BCR) mRNA transcripts with short-read transcriptome profiling of barcoded single-cell libraries generated by droplet-based partitioning. We show that Repertoire and Gene Expression by Sequencing (RAGE-Seq) can generate accurate full-length antigen receptor sequences at nucleotide resolution, infer B-cell clonal evolution and identify alternatively spliced BCR transcripts. We apply RAGE-Seq to 7138 cells sampled from the primary tumor and draining lymph node of a breast cancer patient to track transcriptome profiles of expanded lymphocyte clones across tissues. Our results demonstrate that RAGE-Seq is a powerful method for tracking the clonal evolution from large numbers of lymphocytes applicable to the study of immunity, autoimmunity and cancer.