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.

A single-cell and spatially resolved atlas of human breast cancers.

Cancer-associated fibroblasts (CAFs) found in primary and metastatic tumours are highly versatile, plastic and resilient cells that are actively involved in cancer progression through complex interactions with other cell types in the tumour microenvironment. As well as generating extracellular matrix components that contribute to the structure and function of the tumour stroma, CAFs undergo epigenetic changes to produce secreted factors, exosomes and metabolites that influence tumour angiogenesis, immunology and metabolism. Because of their putative pro-oncogenic functions, CAFs have long been considered an attractive therapeutic target; however, clinical trials of treatment strategies targeting CAFs have mostly ended in failure and, in some cases, accelerated cancer progression and resulted in inferior survival outcomes. Importantly, CAFs are heterogeneous cells and their characteristics and interactions with other cell types might change dynamically as cancers evolve. Studies involving single-cell RNA sequencing and novel mouse models have increased our understanding of CAF diversity, although the context-dependent roles of different CAF populations and their interchangeable plasticity remain largely unknown. Comprehensive characterization of the tumour-promoting and tumour-restraining activities of CAF subtypes, including how these complex bimodal functions evolve and are subjugated by neoplastic cells during cancer progression, might facilitate the development of novel diagnostic and therapeutic approaches. In this Review, the clinical relevance of CAFs is summarized with an emphasis on their value as prognosis factors and therapeutic targets.

Clinical and therapeutic relevance of cancer-associated fibroblasts.

Description Tertiary lymphoid structures in cancer Tertiary lymphoid structures (TLSs) are lymphoid formations that are found in nonlymphoid tissues. TLS can develop in inflamed tissues and are associated with chronic inflammatory disorders, autoimmunity, and cancer. In the setting of tumors, TLSs facilitate the influx of immune cells into the tumor site and have therefore attracted interest as a means of improving anticancer immunity and favorable treatment response in patients. Schumacher and Thommen review the biology of TLSs and outline recent advances in TLS research. They discuss how TLSs are detected and defined, the mechanism(s) of formation in cancer, and the potential of targeting TLSs for therapeutic benefit. —PNK A review discusses tertiary lymphoid structures, ectopic lymphoid aggregates that are associated with tumors but the biology of which is not fully understood. BACKGROUND Tertiary lymphoid structures (TLSs) are organized aggregates of immune cells that form postnatally in nonlymphoid tissues. TLSs are not found under physiological conditions but arise in the context of chronic inflammation, such as in autoimmune disease, chronic infection, and cancer. With few exceptions, the presence of TLSs in tumors correlates with better prognosis and clinical outcome upon immunotherapy, but, in spite of their presumed importance, the drivers of TLS formation in cancer and the contribution of these structures to intratumoral immune responses remain incompletely understood. ADVANCES TLSs resemble secondary lymphoid organs (SLOs) anatomically, and it was originally assumed that their formation would largely be induced by the same stimuli. However, the cell pools and signals that provide inductive stimuli for TLS formation are at least partially different. For instance, several observations suggest that tumor-specific T and B cell immunity may induce some of the molecular factors required for TLS formation and maintenance, and heterogeneity in these drivers may result in distinct TLS states. It has been speculated that TLSs recapitulate SLO functions at the inflamed tissue site, and available evidence suggests that a contribution of TLSs to the strength of tumor-specific immune responses is plausible. However, whether such a contribution primarily involves the boosting of T cell responses generated in SLOs or the development of new T and B cell reactivities remains a key unanswered question. In addition, the presence of TLSs at the tumor site may offer the possibility for the generation of qualitatively distinct immune responses. Specifically, because TLSs are not encapsulated, exposure of TLS-resident immune cells to macromolecules from the inflamed microenvironment appears to be a realistic possibility, and this could potentially sculpt the nature of intratumoral immune responses. Finally, recent studies suggest a role for TLSs in the clinical response to immune checkpoint blockade, which may make these structures attractive therapeutic targets. However, the development of such strategies should take into account the possible consequences of ectopic formation of lymphoid tissue at other body sites. OUTLOOK The prognostic and predictive value of TLSs in cancer has strengthened the interest in these structures as potential mediators of antitumor immunity. Although TLSs have been identified in many cancer types, the markers used to define and characterize TLSs have often varied across studies, complicating efforts to compare predictive value and to assess TLS heterogeneity between cancer types. Thus, the development of standardized approaches to measure TLS number and composition is likely to further reveal their predictive and prognostic value in different disease settings. Related to this, a more comprehensive characterization of TLSs may potentially lead to the identification of a spectrum of TLS states, based on aspects such as cellular composition, location, maturation, and function. Similar to the definition of T cell states in cancer, which has substantially improved our understanding of the role of specific T cell populations in tumor-specific immunity, the molecular definition of TLS states may help to improve their value as prognostic and predictive markers. Finally, a better appreciation of TLS function and the potential contribution of TLSs to autoimmune toxicity will be important to maximize their value as therapeutic targets. Defining TLS states. The molecular definition of TLS states may advance their use as prognostic and predictive markers. Characteristics that will provide insight into the diversity of TLS states include their cellular composition, location, and maturation; properties of their cytokine and chemokine environment; and their B cell receptor (BCR) and T cell receptor (TCR) repertoires. AID, activation-induced cytidine deaminase; CTL, cytotoxic T lymphocyte; FRCs, follicular reticular cells; FDCs, follicular dendritic cells; HEVs, high endothelial venules; Ig, immunoglobulin; TFH, T follicular helper cell; TH, T helper cell; Treg, regulatory T cell. Ectopic lymphoid aggregates, termed tertiary lymphoid structures (TLSs), are formed in numerous cancer types, and, with few exceptions, their presence is associated with superior prognosis and response to immunotherapy. In spite of their presumed importance, the triggers that lead to TLS formation in cancer tissue and the contribution of these structures to intratumoral immune responses remain incompletely understood. Here, we discuss the present knowledge on TLSs in cancer, focusing on (i) the drivers of TLS formation, (ii) the function and contribution of TLSs to the antitumor immune response, and (iii) the potential of TLSs as therapeutic targets in human cancers.

Tertiary lymphoid structures in cancer

Cancer-associated fibroblasts (CAFs) are the key component of tumor stromal. High heterogeneity of CAFs reflects in their origin, phenotype and function. Biological function which can be suggested by biomarkers of distinct CAF subgroups may be different, even opposite, just like water and fire. Identifying CAF subpopulations expressing different biomarkers and reconciling the relationship of the “water and fire” among distinct CAF subsets may be a breakthrough in tumor therapy. Herein, we briefly summarize the biomarkers commonly used or newly identified for distinct CAFs in terms of their features and potential clinical benefits.

/pdf/biomarkers-for-cancer-associated-fibroblasts-vju48kizon.pdf

Biomarkers for cancer-associated fibroblasts.

To examine global changes in breast heterogeneity across different states, we determined the single-cell transcriptomes of > 340,000 cells encompassing normal breast, preneoplastic BRCA1+/- tissue, the major breast cancer subtypes, and pairs of tumors and involved lymph nodes. Elucidation of the normal breast microenvironment revealed striking changes in the stroma of post-menopausal women. Single-cell profiling of 34 treatment-naive primary tumors, including estrogen receptor (ER)+ , HER2+ , and triple-negative breast cancers, revealed comparable diversity among cancer cells and a discrete subset of cycling cells. The transcriptomes of preneoplastic BRCA1+/- tissue versus tumors highlighted global changes in the immune microenvironment. Within the tumor immune landscape, proliferative CD8+ T cells characterized triple-negative and HER2+ cancers but not ER+ tumors, while all subtypes comprised cycling tumor-associated macrophages, thus invoking potentially different immunotherapy targets. Copy number analysis of paired ER+ tumors and lymph nodes indicated seeding by genetically distinct clones or mass migration of primary tumor cells into axillary lymph nodes. This large-scale integration of patient samples provides a high-resolution map of cell diversity in normal and cancerous human breast.

/pdf/a-single-cell-rna-expression-atlas-of-normal-preneoplastic-4grfdun2jp.pdf

A single-cell RNA expression atlas of normal, preneoplastic and tumorigenic states in the human breast.

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.

Chuan En Lam

Papers

Stromal cell diversity associated with immune evasion in human triple-negative breast cancer.