Breast Cancer Immunity: It is TIME for the Next Chapter.

Our ability to interrogate the tumor immune microenvironment (TIME) at an ever-increasing granularity has uncovered critical determinants of disease progression. Not only do we now have a better understanding of the immune response in breast cancer, but it is becoming possible to leverage key mechanisms to effectively combat this disease. Almost every component of the immune system plays a role in enabling or inhibiting breast tumor growth. Building on early seminal work showing the involvement of T cells and macrophages in controlling breast cancer progression and metastasis, single-cell genomics and spatial proteomics approaches have recently expanded our view of the TIME. In this article, we provide a detailed description of the immune response against breast cancer and examine its heterogeneity in disease subtypes. We discuss preclinical models that enable dissecting the mechanisms responsible for tumor clearance or immune evasion and draw parallels and distinctions between human disease and murine counterparts. Last, as the cancer immunology field is moving toward the analysis of the TIME at the cellular and spatial levels, we highlight key studies that revealed previously unappreciated complexity in breast cancer using these technologies. Taken together, this article summarizes what is known in breast cancer immunology through the lens of translational research and identifies future directions to improve clinical outcomes.

ARF6-dependent endocytic trafficking of the Interferon-γ receptor drives adaptive immune resistance in cancer.

Adaptive immune resistance (AIR) is a protective process used by cancer to escape elimination by CD8+ T cells. Inhibition of immune checkpoints PD-1 and CTLA-4 specifically target Interferon-gamma (IFNγ)-driven AIR. AIR begins at the plasma membrane where tumor cell-intrinsic cytokine signaling is initiated. Thus, plasma membrane remodeling by endomembrane trafficking could regulate AIR. Herein we report that the trafficking protein ADP-Ribosylation Factor 6 (ARF6) is critical for IFNγ-driven AIR. ARF6 prevents transport of the receptor to the lysosome, augmenting IFNγR expression, tumor intrinsic IFNγ signaling and downstream expression of immunosuppressive genes. In murine melanoma, loss of ARF6 causes resistance to immune checkpoint blockade (ICB). Likewise, low expression of ARF6 in patient tumors correlates with inferior outcomes with ICB. Our data provide new mechanistic insights into tumor immune escape, defined by ARF6-dependent AIR, and support that ARF6-dependent endomembrane trafficking of the IFNγ receptor influences outcomes of ICB.

Senescence-induced immune remodeling facilitates metastatic adrenal cancer in a sex-dimorphic manner.

Aging markedly increases cancer risk, yet our mechanistic understanding of how aging influences cancer initiation is limited. Here we demonstrate that the loss of ZNRF3, an inhibitor of Wnt signaling that is frequently mutated in adrenocortical carcinoma, leads to the induction of cellular senescence that remodels the tissue microenvironment and ultimately permits metastatic adrenal cancer in old animals. The effects are sexually dimorphic, with males exhibiting earlier senescence activation and a greater innate immune response, driven in part by androgens, resulting in high myeloid cell accumulation and lower incidence of malignancy. Conversely, females present a dampened immune response and increased susceptibility to metastatic cancer. Senescence-recruited myeloid cells become depleted as tumors progress, which is recapitulated in patients in whom a low myeloid signature is associated with worse outcomes. Our study uncovers a role for myeloid cells in restraining adrenal cancer with substantial prognostic value and provides a model for interrogating pleiotropic effects of cellular senescence in cancer.

Interferon Gamma-Inducible NAMPT in Melanoma Cells Serves as a Mechanism of Resistance to Enhance Tumor Growth.

(1) Background: Immune cells infiltrate the tumor microenvironment and secrete inflammatory cytokines, including interferons (IFNs), to drive antitumor responses and promote tumor clearance. However, recent evidence suggests that sometimes, tumor cells can also harness IFNs to enhance growth and survival. The essential NAD+ salvage pathway enzyme nicotinamide phosphoribosyltransferase (NAMPT) gene is constitutively expressed in cells during normal homeostasis. However, melanoma cells have higher energetic demands and elevated NAMPT expression. We hypothesized that interferon gamma (IFNγ) regulates NAMPT in tumor cells as a mechanism of resistance that impedes the normal anti-tumorigenic effects of IFNγ. (2) Methods: Utilizing a variety of melanoma cells, mouse models, Crispr-Cas9, and molecular biology techniques, we explored the importance of IFNγ-inducible NAMPT during melanoma growth. (3) Results: We demonstrated that IFNγ mediates the metabolic reprogramming of melanoma cells by inducing Nampt through a Stat1 binding site in the Nampt gene, increasing cell proliferation and survival. Further, IFN/STAT1-inducible Nampt promotes melanoma in vivo. (4) Conclusions: We provided evidence that melanoma cells directly respond to IFNγ by increasing NAMPT levels, improving their fitness and growth in vivo (control n = 36, SBS KO n = 46). This discovery unveils a possible therapeutic target that may improve the efficacy of immunotherapies involving IFN responses in the clinic.

Epithelial-myeloid exchange of MHC class II constrains immunity and microbiota composition.

Intestinal epithelial cells (IECs) have long been understood to express high levels of major histocompatibility complex class II (MHC class II) molecules but are not considered canonical antigen-presenting cells, and the impact of IEC-MHC class II signaling on gut homeostasis remains enigmatic. As IECs serve as the primary barrier between underlying host immune cells, we reasoned that IEC-intrinsic antigen presentation may play a role in responses toward the microbiota. Mice with an IEC-intrinsic deletion of MHC class II (IECΔMHC class II) are healthy but have fewer microbial-bound IgA, regulatory T cells (Tregs), and immune repertoire selection. This was associated with increased interindividual microbiota variation and altered proportions of two taxa in the ileum where MHC class II on IECs is highest. Intestinal mononuclear phagocytes (MNPs) have similar MHC class II transcription but less surface MHC class II and are capable of acquiring MHC class II from IECs. Thus, epithelial-myeloid interactions mediate development of adaptive responses to microbial antigens within the gastrointestinal tract.

An immune-humanized patient-derived xenograft model of estrogen-independent, hormone receptor positive metastatic breast cancer.

BACKGROUND: Metastatic breast cancer (MBC) is incurable, with a 5-year survival rate of 28%. In the USA, more than 42,000 patients die from MBC every year. The most common type of breast cancer is estrogen receptor-positive (ER+), and more patients die from ER+ breast cancer than from any other subtype. ER+ tumors can be successfully treated with hormone therapy, but many tumors acquire endocrine resistance, at which point treatment options are limited. There is an urgent need for model systems that better represent human ER+ MBC in vivo, where tumors can metastasize. Patient-derived xenografts (PDX) made from MBC spontaneously metastasize, but the immunodeficient host is a caveat, given the known role of the immune system in tumor progression and response to therapy. Thus, we attempted to develop an immune-humanized PDX model of ER+ MBC. METHODS: NSG-SGM3 mice were immune-humanized with CD34+ hematopoietic stem cells, followed by engraftment of human ER+ endocrine resistant MBC tumor fragments. Strategies for exogenous estrogen supplementation were compared, and immune-humanization in blood, bone marrow, spleen, and tumors was assessed by flow cytometry and tissue immunostaining. Characterization of the new model includes assessment of the human tumor microenvironment performed by immunostaining. RESULTS: We describe the development of an immune-humanized PDX model of estrogen-independent endocrine resistant ER+ MBC. Importantly, our model harbors a naturally occurring ESR1 mutation, and immune-humanization recapitulates the lymphocyte-excluded and myeloid-rich tumor microenvironment of human ER+ breast tumors. CONCLUSION: This model sets the stage for development of other clinically relevant models of human breast cancer and should allow future studies on mechanisms of endocrine resistance and tumor-immune interactions in an immune-humanized in vivo setting.

Disrupted macrophage metabolic reprogramming in aged soleus muscle during early recovery following disuse atrophy.

Aged skeletal muscle is characterized by poor muscle recovery following disuse coinciding with an impaired muscle pro-inflammatory macrophage response. Macrophage inflammatory status is regulated by its metabolic state, but little is understood of macrophage metabolism and its relation to macrophage inflammation in the context of muscle recovery and aging. Therefore, the purpose of this study was to thoroughly characterize macrophage metabolism and inflammation in aged muscle during early recovery following disuse atrophy using single cell transcriptomics and functional assays. Young (4-5 months) and old (20-22 months) male C57BL/6 mice underwent 14 days of hindlimb unloading followed by 4 days of ambulatory recovery. CD45+ cells were isolated from solei muscles and analyzed using 10x Genomics single cell RNA sequencing. We found that aged pro-inflammatory macrophage clusters were characterized with an impaired inflammatory and glycolytic transcriptome, and this dysregulation was accompanied by a suppression of HIF-1α and its immediate downstream target, Glut1. As a follow-up, bone marrow-derived macrophages were isolated from a separate cohort of young and old mice at 4-d recovery and were polarized to a pro-inflammatory phenotype and used for glycolysis stress test, phagocytosis activity assay, and targeted GC-MS metabolomics. Aged bone marrow-derived pro-inflammatory macrophages were characterized with impaired glycolysis and phagocytosis function, decreased succinate and an accumulation of glycolytic metabolic intermediates overall supporting reduced glycolytic flux and macrophage function. Our results indicate that the metabolic reprograming and function of aged skeletal muscle pro-inflammatory macrophages are dysfunctional during early recovery from disuse atrophy possibly attributing to attenuated regrowth.

Blocking Short-Form Ron Eliminates Breast Cancer Metastases through Accumulation of Stem-Like CD4+ T Cells That Subvert Immunosuppression.

Immunotherapy has potential to prevent and treat metastatic breast cancer, but strategies to enhance immune-mediated killing of metastatic tumors are urgently needed. We report that a ligand-independent isoform of Ron kinase (SF-Ron) is a key target to enhance immune infiltration and eradicate metastatic tumors. Host-specific deletion of SF-Ron caused recruitment of lymphocytes to micrometastases, augmented tumor-specific T-cell responses, and nearly eliminated breast cancer metastasis in mice. Lack of host SF-Ron caused stem-like TCF1+ CD4+ T cells with type I differentiation potential to accumulate in metastases and prevent metastatic outgrowth. There was a corresponding increase in tumor-specific CD8+ T cells, which were also required to eliminate lung metastases. Treatment of mice with a Ron kinase inhibitor increased tumor-specific CD8+ T cells and protected from metastatic outgrowth. These data provide a strong preclinical rationale to pursue small-molecule Ron kinase inhibitors for the prevention and treatment of metastatic breast cancer. SIGNIFICANCE: The discovery that SF-Ron promotes antitumor immune responses has significant clinical implications. Therapeutic antibodies targeting full-length Ron may not be effective for immunotherapy; poor efficacy of such antibodies in trials may be due to their inability to block SF-Ron. Our data warrant trials with inhibitors targeting SF-Ron in combination with immunotherapy. This article is highlighted in the In This Issue feature, p. 2945.

A Stat1 bound enhancer promotes Nampt expression and function within tumor associated macrophages.

Tumor associated macrophage responses are regulated by distinct metabolic states that affect their function. However, the ability of specific signals in the local tumor microenvironment to program macrophage metabolism remains under investigation. Here, we identify NAMPT, the rate limiting enzyme in NAD salvage synthesis, as a target of STAT1 during cellular activation by interferon gamma, an important driver of macrophage polarization and antitumor responses. We demonstrate that STAT1 occupies a conserved element within the first intron of Nampt, termed Nampt-Regulatory Element-1 (NRE1). Through disruption of NRE1 or pharmacological inhibition, a subset of M1 genes is sensitive to NAMPT activity through its impact on glycolytic processes. scRNAseq is used to profile in vivo responses by NRE1-deficient, tumor-associated leukocytes in melanoma tumors through the creation of a unique mouse strain. Reduced Nampt and inflammatory gene expression are present in specific myeloid and APC populations; moreover, targeted ablation of NRE1 in macrophage lineages results in greater tumor burden. Finally, elevated NAMPT expression correlates with IFNγ responses and melanoma patient survival. This study identifies IFN and STAT1-inducible Nampt as an important factor that shapes the metabolic program and function of tumor associated macrophages.

CIPR: a web-based R/shiny app and R package to annotate cell clusters in single cell RNA sequencing experiments.

BACKGROUND: Single cell RNA sequencing (scRNAseq) has provided invaluable insights into cellular heterogeneity and functional states in health and disease. During the analysis of scRNAseq data, annotating the biological identity of cell clusters is an important step before downstream analyses and it remains technically challenging. The current solutions for annotating single cell clusters generally lack a graphical user interface, can be computationally intensive or have a limited scope. On the other hand, manually annotating single cell clusters by examining the expression of marker genes can be subjective and labor-intensive. To improve the quality and efficiency of annotating cell clusters in scRNAseq data, we present a web-based R/Shiny app and R package, Cluster Identity PRedictor (CIPR), which provides a graphical user interface to quickly score gene expression profiles of unknown cell clusters against mouse or human references, or a custom dataset provided by the user. CIPR can be easily integrated into the current pipelines to facilitate scRNAseq data analysis. RESULTS: CIPR employs multiple approaches for calculating the identity score at the cluster level and can accept inputs generated by popular scRNAseq analysis software. CIPR provides 2 mouse and 5 human reference datasets, and its pipeline allows inter-species comparisons and the ability to upload a custom reference dataset for specialized studies. The option to filter out lowly variable genes and to exclude irrelevant reference cell subsets from the analysis can improve the discriminatory power of CIPR suggesting that it can be tailored to different experimental contexts. Benchmarking CIPR against existing functionally similar software revealed that our algorithm is less computationally demanding, it performs significantly faster and provides accurate predictions for multiple cell clusters in a scRNAseq experiment involving tumor-infiltrating immune cells. CONCLUSIONS: CIPR facilitates scRNAseq data analysis by annotating unknown cell clusters in an objective and efficient manner. Platform independence owing to Shiny framework and the requirement for a minimal programming experience allows this software to be used by researchers from different backgrounds. CIPR can accurately predict the identity of a variety of cell clusters and can be used in various experimental contexts across a broad spectrum of research areas.

Microglia influence host defense, disease, and repair following murine coronavirus infection of the central nervous system.

The present study examines functional contributions of microglia in host defense, demyelination, and remyelination following infection of susceptible mice with a neurotropic coronavirus. Treatment with PLX5622, an inhibitor of colony stimulating factor 1 receptor (CSF1R) that efficiently depletes microglia, prior to infection of the central nervous system (CNS) with the neurotropic JHM strain of mouse hepatitis virus (JHMV) resulted in increased mortality compared with control mice that correlated with impaired control of viral replication. Single cell RNA sequencing (scRNASeq) of CD45+ cells isolated from the CNS revealed that PLX5622 treatment resulted in muted CD4+ T cell activation profile that was associated with decreased expression of transcripts encoding MHC class II and CD86 in macrophages but not dendritic cells. Evaluation of spinal cord demyelination revealed a marked increase in white matter damage in PLX5622-treated mice that corresponded with elevated expression of transcripts encoding disease-associated proteins Osteopontin (Spp1), Apolipoprotein E (Apoe), and Triggering receptor expressed on myeloid cells 2 (Trem2) that were enriched within macrophages. In addition, PLX5622 treatment dampened expression of Cystatin F (Cst7), Insulin growth factor 1 (Igf1), and lipoprotein lipase (Lpl) within macrophage populations which have been implicated in promoting repair of damaged nerve tissue and this was associated with impaired remyelination. Collectively, these findings argue that microglia tailor the CNS microenvironment to enhance control of coronavirus replication as well as dampen the severity of demyelination and influence repair.

Mitochondrial Pyruvate Carrier 1 Promotes Peripheral T Cell Homeostasis through Metabolic Regulation of Thymic Development.

Metabolic pathways regulate T cell development and function, but many remain understudied. Recently, the mitochondrial pyruvate carrier (MPC) was identified as the transporter that mediates pyruvate entry into mitochondria, promoting pyruvate oxidation. Here we find that deleting Mpc1, an obligate MPC subunit, in the hematopoietic system results in a specific reduction in peripheral αß T cell numbers. MPC1-deficient T cells have defective thymic development at the ß-selection, intermediate single positive (ISP)-to-double-positive (DP), and positive selection steps. We find that early thymocytes deficient in MPC1 display alterations to multiple pathways involved in T cell development. This results in preferred escape of more activated T cells. Finally, mice with hematopoietic deletion of Mpc1 are more susceptible to experimental autoimmune encephalomyelitis. Altogether, our study demonstrates that pyruvate oxidation by T cell precursors is necessary for optimal αß T cell development and that its deficiency results in reduced but activated peripheral T cell populations.

Inhibition of SHP-1 Expands the Repertoire of Antitumor T Cells Available to Respond to Immune Checkpoint Blockade.

The presence and activity of CD8+ T cells within the tumor microenvironment are essential for the control of tumor growth. Utilizing B16-F10 melanoma tumors that express altered peptide ligands of chicken ovalbumin, OVA257-264, we measured high- and low-affinity OVA-specific responses following adoptive transfer of OT-I CD8+ T cell into mice subsequently challenged with tumors. T-cell receptor (TCR) affinity positively correlated with the frequency of OT-I tumor-infiltrating lymphocytes (TIL). Differences in TCR affinity inversely corresponded to in vivo tumor growth rate. Blockade of the PD-1 and CTLA-4 checkpoints preferentially increased the frequency and antitumor function of TIL responding to high-affinity antigens, while failing to enhance the antitumor activity of low-affinity T cells. To determine whether lowering the TCR activation threshold could enhance the breadth and magnitude of the antitumor T-cell response, we inhibited Src homology region 2 domain-containing phosphatase 1 (SHP-1) in OT-I T cells prior to tumor antigen exposure. SHP-1 knockdown increased the cytokine-producing potential of high- and low-affinity T cells but failed to enhance control of tumor growth. In contrast, when SHP-1 knockdown of OT-I T cells was combined with immunotherapy, we observed a significant and long-lasting suppression of tumor growth mediated by low-affinity T cells. We conclude that lowering the TCR activation threshold by targeting SHP-1 expands the repertoire of T cells available to respond to conventional checkpoint blockade, leading to enhanced control of tumor growth.

MicroRNA-155 coordinates the immunological landscape within murine melanoma and correlates with immunity in human cancers.

miR-155 has recently emerged as an important promoter of antitumor immunity through its functions in T lymphocytes. However, the impact of T cell-expressed miR-155 on immune cell dynamics in solid tumors remains unclear. In the present study, we used single-cell RNA sequencing to define the CD45+ immune cell populations at different time points within B16F10 murine melanoma tumors growing in either wild-type or miR-155 T cell conditional knockout (TCKO) mice. miR-155 was required for optimal T cell activation and reinforced the T cell response at the expense of infiltrating myeloid cells. Further, myeloid cells from tumors growing in TCKO mice were defined by an increase in wound healing genes and a decreased IFN-γ-response gene signature. Finally, we found that miR-155 expression predicted a favorable outcome in human melanoma patients and was associated with a strong immune signature. Moreover, gene expression analysis of The Cancer Genome Atlas (TCGA) data revealed that miR-155 expression also correlates with an immune-enriched subtype in 29 other human solid tumors. Together, our study provides an unprecedented analysis of the cell types and gene expression signatures of immune cells within experimental melanoma tumors and elucidates the role of miR-155 in coordinating antitumor immune responses in mammalian tumors.