Spectral Flow Cytometry Methods and Pipelines for Comprehensive Immunoprofiling of Human Peripheral Blood and Bone Marrow.

Profiling hematopoietic and immune cells provides important information about disease risk, disease status, and therapeutic responses. Spectral flow cytometry enables high-dimensional single-cell evaluation of large cohorts in a high-throughput manner. Here, we designed, optimized, and implemented new methods for deep immunophenotyping of human peripheral blood and bone marrow by spectral flow cytometry. Two blood antibody panels capture 48 cell-surface markers to assess more than 58 cell phenotypes, including subsets of T cells, B cells, monocytes, natural killer (NK) cells, and dendritic cells, and their respective markers of exhaustion, activation, and differentiation in less than 2 mL of blood. A bone marrow antibody panel captures 32 markers for 35 cell phenotypes, including stem/progenitor populations, T-cell subsets, dendritic cells, NK cells, and myeloid cells in a single tube. We adapted and developed innovative flow cytometric analysis algorithms, originally developed for single-cell genomics, to improve data integration and visualization. We also highlight technical considerations for users to ensure data fidelity. Our protocol and analysis pipeline accurately identifies rare cell types, discerns differences in cell abundance and phenotype across donors, and shows concordant immune landscape trends in patients with known hematologic malignancy. SIGNIFICANCE: This study introduces optimized methods and analysis algorithms that enhance capabilities in comprehensive immunophenotyping of human blood and bone marrow using spectral flow cytometry. This approach facilitates detection of rare cell types, enables measurement of cell variations across donors, and provides proof-of-concept in identifying known hematologic malignancies. By unlocking complexities of hematopoietic and immune landscapes at the single-cell level, this advancement holds potential for understanding disease states and therapeutic responses.

Clonal hematopoiesis in older patients with breast cancer receiving chemotherapy.

BACKGROUND: The expansion of hematopoietic stem cells carrying recurrent somatic mutations, termed clonal hematopoiesis (CH), is common in elderly individuals and is associated with increased risk of myeloid malignancy and all-cause mortality. Though chemotherapy is a known risk factor for developing CH, how myelosuppressive therapies affect the short-term dynamics of CH remains incompletely understood. Most studies have been limited by retrospective design, heterogeneous patient populations, varied techniques to identifying CH, and analysis of single timepoints. METHODS: We examined serial samples from 40 older women with triple-negative or hormone receptor-positive breast cancer treated on the prospective ADjuVANt Chemotherapy in the Elderly trial to evaluate the prevalence and dynamics of CH at baseline and throughout chemotherapy (6 and 12 weeks). RESULTS: CH was detected in 44% of patients at baseline and in 53% at any timepoint. Baseline patient characteristics were not associated with CH. Over the course of treatment, mutations exhibited a variety of dynamics, including emergence, expansion, contraction, and disappearance. All mutations in TP53 (n = 3) and PPM1D (n = 4), genes that regulate the DNA damage response, either became detectable or expanded over the course of treatment. Neutropenia was more common in patients with CH, particularly when the mutations became detectable during treatment, and CH was significantly associated with cyclophosphamide dose reductions and holds (P = .02). CONCLUSIONS: Our study shows that CH is common, dynamic, and of potential clinical significance in this population. Our results should stimulate larger efforts to understand the biological and clinical importance of CH in solid tumor malignancies. TRIAL REGISTRATION: ClinicalTrials.gov (https://clinicaltrials.gov/ct2/show/NCT03858322). Clinical trial registration number: NCT03858322.

Challenges and opportunities for modeling aging and cancer.

Age is among the main risk factors for cancer, and any cancer study in adults is faced with an aging tissue and organism. Yet, pre-clinical studies are carried out using young mice and are not able to address the impact of aging and associated comorbidities on disease biology and treatment outcomes. Here, we discuss the limitations of current mouse cancer models and suggest strategies for developing novel models to address these major gaps in knowledge and experimental approaches.

'ADVANCE' (a pilot trial) ADjuVANt chemotherapy in the elderly: Developing and evaluating lower-toxicity chemotherapy options for older patients with breast cancer.

INTRODUCTION: Older adults with breast cancer receiving neo/adjuvant chemotherapy are at high risk for poor outcomes and are underrepresented in clinical trials. The ADVANCE (ADjuVANt Chemotherapy in the Elderly) trial evaluated the feasibility of two neo/adjuvant chemotherapy regimens in parallel-enrolling cohorts of older patients with human epidermal growth factor receptor 2-negative breast cancer: cohort 1-triple-negative; cohort 2-hormone receptor-positive. MATERIALS AND METHODS: Adults age ≥ 70 years with stage I-III breast cancer warranting neo/adjuvant chemotherapy were enrolled. Cohort 1 received weekly carboplatin (area under the curve 2) and weekly paclitaxel 80 mg/m2 for twelve weeks; cohort 2 received weekly paclitaxel 80 mg/m2 plus every-three-weekly cyclophosphamide 600 mg/m2 over twelve weeks. The primary study endpoint was feasibility, defined as ≥80% of patients receiving ≥80% of intended weeks/doses of therapy. All dose modifications were applied per clinician discretion. RESULTS: Forty women (n = 20 per cohort) were enrolled from March 25, 2019 through August 3, 2020 from three centers; 45% and 35% of patients in cohorts 1 and 2 were age > 75, respectively. Neither cohort achieved targeted thresholds for feasibility. In cohort 1, eight (40.0%) met feasibility (95% confidence interval [CI] = 19.1-63.9%), while ten (50.0%) met feasibility in cohort 2 (95% CI = 27.2-72.8). Neutropenia was the most common grade 3-4 toxicity (cohort 1-65%, cohort 2-55%). In cohort 1, 80% and 85% required ≥1 dose holds of carboplatin and/or paclitaxel, respectively. In cohort 2, 10% required dose hold(s) for cyclophosphamide and/or 65% for paclitaxel. DISCUSSION: In this pragmatic pilot examining chemotherapy regimens in older adults with breast cancer, neither regimen met target goals for feasibility. Developing efficacious and tolerable regimens for older patients with breast cancer who need chemotherapy remains an important goal. CLINICALTRIALS: gov Identifier: NCT03858322.

Pro-inflammatory megakaryocyte gene expression in murine models of breast cancer.

Despite abundant research demonstrating that platelets can promote tumor cell metastasis, whether primary tumors affect platelet-producing megakaryocytes remains understudied. In this study, we used a spontaneous murine model of breast cancer to show that tumor burden reduced megakaryocyte number and size and disrupted polyploidization. Single-cell RNA sequencing demonstrated that megakaryocytes from tumor-bearing mice exhibit a pro-inflammatory phenotype, epitomized by increased Ctsg, Lcn2, S100a8, and S100a9 transcripts. Protein S100A8/A9 and lipocalin-2 levels were also increased in platelets, suggesting that tumor-induced alterations to megakaryocytes are passed on to their platelet progeny, which promoted in vitro tumor cell invasion and tumor cell lung colonization to a greater extent than platelets from wild-type animals. Our study is the first to demonstrate breast cancer-induced alterations in megakaryocytes, leading to qualitative changes in platelet content that may feedback to promote tumor metastasis.

Clonal barcoding with qPCR detection enables live cell functional analyses for cancer research.

Single-cell analysis methods are valuable tools; however, current approaches do not easily enable live cell retrieval. That is a particular issue when further study of cells that were eliminated during experimentation could provide critical information. We report a clonal molecular barcoding method, called SunCatcher, that enables longitudinal tracking and live cell functional analysis. From complex cell populations, we generate single cell-derived clonal populations, infect each with a unique molecular barcode, and retain stocks of individual barcoded clones (BCs). We develop quantitative PCR-based and next-generation sequencing methods that we employ to identify and quantify BCs in vitro and in vivo. We apply SunCatcher to various breast cancer cell lines and combine respective BCs to create versions of the original cell lines. While the heterogeneous BC pools reproduce their original parental cell line proliferation and tumor progression rates, individual BCs are phenotypically and functionally diverse. Early spontaneous metastases can also be identified and quantified. SunCatcher thus provides a rapid and sensitive approach for studying live single-cell clones and clonal evolution, and performing functional analyses.

Platelets upregulate tumor cell programmed death ligand 1 in an epidermal growth factor receptor-dependent manner in vitro.

Programmed death ligand 1 (PD-L1) is an immune checkpoint protein that suppresses cytotoxic T lymphocytes and is often overexpressed in cancers. Due to favorable clinical trial results, immune checkpoint inhibition (ICI) is part of Food and Drug Administration approved immuno-oncology therapies; however, not all patients benefit from ICI therapy. High blood platelet-to-lymphocyte ratio has been associated with failure of ICI treatment, but whether platelets have a role in hindering ICI response is unclear. Here, we report that coculturing platelets with cancer cell lines increased protein and gene expression of tumor cell PD-L1, which was reduced by antiplatelet agents, such as aspirin and ticagrelor. Platelet cytokine arrays revealed that the well-established cytokines, including interferon-γ, were not the main regulators of platelet-mediated PD-L1 upregulation. Instead, the high molecular weight epidermal growth factor (EGF) is abundant in platelets, which caused an upregulation of tumor cell PD-L1. Both an EGF-neutralizing antibody and cetuximab (EGF receptor [EGFR] monoclonal antibody) inhibited platelet-induced increases in tumor cell PD-L1, suggesting that platelets induce tumor cell PD-L1 in an EGFR-dependent manner. Our data reveal a novel mechanism for platelets in tumor immune escape and warrant further investigation to determine if targeting platelets improves ICI therapeutic responses.

Understanding resistance to immune checkpoint inhibitors in advanced breast cancer.

INTRODUCTION: The addition of immune checkpoint inhibitors (ICIs) to frontline chemotherapy has improved survival for patients with advanced triple-negative breast cancer (TNBC) expressing programmed death-ligand 1 (PD-L1). Nonetheless, most patients develop resistance, with outcomes remaining poor for this population. Moreover, unsatisfactory activity has been observed with ICIs in PD-L1-negative TNBC and in other breast cancer (BC) subtypes, warranting a deeper understanding of resistance to ICIs in BC. AREAS COVERED: We discuss the immune landscape of distinct BC subtypes, review the clinical activity of immunotherapy in BC, and highlight strategies under development to overcome resistance to ICIs. EXPERT OPINION: Activity and resistance to ICIs in BC are strongly related to the intrinsic immunophenotype of the tumor tissue. Several promising biomarkers reflecting the immunological state of BC are emerging, with only PD-L1 expression currently adopted into clinical practice. However, limitations make of PD-L1 a sub-optimal biomarker for patient selection, which require efforts to integrate this marker with other immunological features. Concomitantly, a wide variety of drug combinations designed to overcome immune-resistance are being evaluated, with some encouraging signals observed in early-phase trials. Combination strategies tailored to patient and tumor immunophenotype may allow to overcome resistance and fully exploit the potential of ICIs.

The Immunology of Hormone Receptor Positive Breast Cancer.

Immune checkpoint blockade (ICB) has revolutionized the treatment of cancer patients. The main focus of ICB has been on reinvigorating the adaptive immune response, namely, activating cytotoxic T cells. ICB has demonstrated only modest benefit against advanced breast cancer, as breast tumors typically establish an immune suppressive tumor microenvironment (TME). Triple-negative breast cancer (TNBC) is associated with infiltration of tumor infiltrating lymphocytes (TILs) and patients with TNBC have shown clinical responses to ICB. In contrast, hormone receptor positive (HR+) breast cancer is characterized by low TIL infiltration and minimal response to ICB. Here we review how HR+ breast tumors establish a TME devoid of TILs, have low HLA class I expression, and recruit immune cells, other than T cells, which impact response to therapy. In addition, we review emerging technologies that have been employed to characterize components of the TME to reveal that tumor associated macrophages (TAMs) are abundant in HR+ cancer, are highly immune-suppressive, associated with tumor progression, chemotherapy and ICB-resistance, metastasis and poor survival. We reveal novel therapeutic targets and possible combinations with ICB to enhance anti-tumor immune responses, which may have great potential in HR+ breast cancer.

Subclonal cooperation drives metastasis by modulating local and systemic immune microenvironments.

Most human tumours are heterogeneous, composed of cellular clones with different properties present at variable frequencies. Highly heterogeneous tumours have poor clinical outcomes, yet the underlying mechanism remains poorly understood. Here, we show that minor subclones of breast cancer cells expressing IL11 and FIGF (VEGFD) cooperate to promote metastatic progression and generate polyclonal metastases composed of driver and neutral subclones. Expression profiling of the epithelial and stromal compartments of monoclonal and polyclonal primary and metastatic lesions revealed that this cooperation is indirect, mediated through the local and systemic microenvironments. We identified neutrophils as a leukocyte population stimulated by the IL11-expressing minor subclone and showed that the depletion of neutrophils prevents metastatic outgrowth. Single-cell RNA-seq of CD45+ cell populations from primary tumours, blood and lungs demonstrated that IL11 acts on bone-marrow-derived mesenchymal stromal cells, which induce pro-tumorigenic and pro-metastatic neutrophils. Our results indicate key roles for non-cell-autonomous drivers and minor subclones in metastasis.

Metastasis as a systemic disease: molecular insights and clinical implications.

Metastasis is a complex systemic disease that develops as a result of interactions between tumor cells and their local and distant microenvironments. Local and systemic immune-related changes play especially critical roles in limiting or enabling the development of metastatic disease. Although anti-tumor immune responses likely eliminate most early primary and metastatic lesions, factors secreted by cancer or stromal cells in the primary tumor can mobilize and activate cells in distant organs in a way that promotes the outgrowth of disseminated cancer cells into macrometastatic lesions. Therefore, the prevention, detection, and effective treatment of metastatic disease require a deeper understanding of the systemic effects of primary tumors as well as predisposing hereditary and acquired host factors including chronic inflammatory conditions. The success of immunotherapy in a subset of cancer patients is an example of how modulating the microenvironment and tumor-immune cell interactions can be exploited for the effective eradiation of even advanced-stage tumors. Here, we highlight emerging insights and clinical implications of cancer as a systemic disease.

Interferon Signaling Is Diminished with Age and Is Associated with Immune Checkpoint Blockade Efficacy in Triple-Negative Breast Cancer.

Immune checkpoint blockade (ICB) therapy, which targets T cell-inhibitory receptors, has revolutionized cancer treatment. Among the breast cancer subtypes, evaluation of ICB has been of greatest interest in triple-negative breast cancer (TNBC) due to its immunogenicity, as evidenced by the presence of tumor-infiltrating lymphocytes and elevated PD-L1 expression relative to other subtypes. TNBC incidence is equally distributed across the age spectrum, affecting 10% to 15% of women in all age groups. Here we report that increased immune dysfunction with age limits ICB efficacy in aged TNBC-bearing mice. The tumor microenvironment in both aged mice and patients with TNBC shows decreased IFN signaling and antigen presentation, suggesting failed innate immune activation with age. Triggering innate immune priming with a STING agonist restored response to ICB in aged mice. Our data implicate age-related immune dysfunction as a mechanism of ICB resistance in mice and suggest potential prognostic utility of assessing IFN-related genes in patients with TNBC receiving ICB therapy. SIGNIFICANCE: These data demonstrate for the first time that age determines the T cell-inflamed phenotype in TNBC and affects response to ICB in mice. Evaluating IFN-related genes from tumor genomic data may aid identification of patients for whom combination therapy including an IFN pathway activator with ICB may be required.This article is highlighted in the In This Issue feature, p. 1143.

CDK4/6 Inhibition in Cancer: Beyond Cell Cycle Arrest.

Pharmacologic inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) have recently entered the therapeutic armamentarium of clinical oncologists, and show promising activity in patients with breast and other cancers. Although their chief mechanism of action is inhibition of retinoblastoma (RB) protein phosphorylation and thus the induction of cell cycle arrest, CDK4/6 inhibitors alter cancer cell biology in other ways that can also be leveraged for therapeutic benefit. These include modulation of mitogenic kinase signaling, induction of a senescence-like phenotype, and enhancement of cancer cell immunogenicity. We describe here the less-appreciated effects of CDK4/6 inhibitors on cancer cells, and suggest ways by which they might be exploited to enhance the benefits of these agents for cancer patients.

Modulating Bone Marrow Hematopoietic Lineage Potential to Prevent Bone Metastasis in Breast Cancer.

The presence of disseminated tumor cells in breast cancer patient bone marrow aspirates predicts decreased recurrence-free survival. Although it is appreciated that physiologic, pathologic, and therapeutic conditions impact hematopoiesis, it remains unclear whether targeting hematopoiesis presents opportunities for limiting bone metastasis. Using preclinical breast cancer models, we discovered that marrow from mice treated with the bisphosphonate zoledronic acid (ZA) are metastasis-suppressive. Specifically, ZA modulated hematopoietic myeloid/osteoclast progenitor cell (M/OCP) lineage potential to activate metastasis-suppressive activity. Granulocyte-colony stimulating factor (G-CSF) promoted ZA resistance by redirecting M/OCP differentiation. We identified M/OCP and bone marrow transcriptional programs associated with metastasis suppression and ZA resistance. Analysis of patient blood samples taken at randomization revealed that women with high-plasma G-CSF experienced significantly worse outcome with adjuvant ZA than those with lower G-CSF levels. Our findings support discovery of therapeutic strategies to direct M/OCP lineage potential and biomarkers that stratify responses in patients at risk of recurrence.Significance: Bone marrow myeloid/osteoclast progenitor cell lineage potential has a profound impact on breast cancer bone metastasis and can be modulated by G-CSF and bone-targeting agents. Cancer Res; 78(18); 5300-14. ©2018 AACR.

IL-1ß inflammatory response driven by primary breast cancer prevents metastasis-initiating cell colonization.

Lack of insight into mechanisms governing breast cancer metastasis has precluded the development of curative therapies. Metastasis-initiating cancer cells (MICs) are uniquely equipped to establish metastases, causing recurrence and therapeutic resistance. Using various metastasis models, we discovered that certain primary tumours elicit a systemic inflammatory response involving interleukin-1ß (IL-1ß)-expressing innate immune cells that infiltrate distant MIC microenvironments. At the metastatic site, IL-1ß maintains MICs in a ZEB1-positive differentiation state, preventing MICs from generating highly proliferative E-cadherin-positive progeny. Thus, when the inherent plasticity of MICs is impeded, overt metastases cannot be established. Ablation of the pro-inflammatory response or inhibition of the IL-1 receptor relieves the differentiation block and results in metastatic colonization. Among patients with lymph node-positive breast cancer, high primary tumour IL-1ß expression is associated with better overall survival and distant metastasis-free survival. Our data reveal complex interactions that occur between primary tumours and disseminated MICs that could be exploited to improve patient survival.

Inhibition of epithelial cell migration and Src/FAK signaling by SIRT3.

Metastasis remains the leading cause of cancer mortality, and reactive oxygen species (ROS) signaling promotes the metastatic cascade. However, the molecular pathways that control ROS signaling relevant to metastasis are little studied. Here, we identify SIRT3, a mitochondrial deacetylase, as a regulator of cell migration via its control of ROS signaling. We find that, although mitochondria are present at the leading edge of migrating cells, SIRT3 expression is down-regulated during migration, resulting in elevated ROS levels. This SIRT3-mediated control of ROS represses Src oxidation and attenuates focal adhesion kinase (FAK) activation. SIRT3 overexpression inhibits migration and metastasis in breast cancer cells. Finally, in human breast cancers, SIRT3 expression is inversely correlated with metastatic outcome and Src/FAK signaling. Our results reveal a role for SIRT3 in cell migration, with important implications for breast cancer progression.

Accounting for tumor heterogeneity when using CRISPR-Cas9 for cancer progression and drug sensitivity studies.

Gene editing protocols often require the use of a subcloning step to isolate successfully edited cells, the behavior of which is then compared to the aggregate parental population and/or other non-edited subclones. Here we demonstrate that the inherent functional heterogeneity present in many cell lines can render these populations inappropriate controls, resulting in erroneous interpretations of experimental findings. We describe a novel CRISPR/Cas9 protocol that incorporates a single-cell cloning step prior to gene editing, allowing for the generation of appropriately matched, functionally equivalent control and edited cell lines. As a proof of concept, we generated matched control and osteopontin-knockout Her2+ and Estrogen receptor-negative murine mammary carcinoma cell lines and demonstrated that the osteopontin-knockout cell lines exhibit the expected biological phenotypes, including unaffected primary tumor growth kinetics and reduced metastatic outgrowth in female FVB mice. Using these matched cell lines, we discovered that osteopontin-knockout mammary tumors were more sensitive than control tumors to chemotherapy in vivo. Our results demonstrate that heterogeneity must be considered during experimental design when utilizing gene editing protocols and provide a solution to account for it.

CDK4/6 inhibition triggers anti-tumour immunity.

Cyclin-dependent kinases 4 and 6 (CDK4/6) are fundamental drivers of the cell cycle and are required for the initiation and progression of various malignancies. Pharmacological inhibitors of CDK4/6 have shown significant activity against several solid tumours. Their primary mechanism of action is thought to be the inhibition of phosphorylation of the retinoblastoma tumour suppressor, inducing G1 cell cycle arrest in tumour cells. Here we use mouse models of breast carcinoma and other solid tumours to show that selective CDK4/6 inhibitors not only induce tumour cell cycle arrest, but also promote anti-tumour immunity. We confirm this phenomenon through transcriptomic analysis of serial biopsies from a clinical trial of CDK4/6 inhibitor treatment for breast cancer. The enhanced anti-tumour immune response has two underpinnings. First, CDK4/6 inhibitors activate tumour cell expression of endogenous retroviral elements, thus increasing intracellular levels of double-stranded RNA. This in turn stimulates production of type III interferons and hence enhances tumour antigen presentation. Second, CDK4/6 inhibitors markedly suppress the proliferation of regulatory T cells. Mechanistically, the effects of CDK4/6 inhibitors both on tumour cells and on regulatory T cells are associated with reduced activity of the E2F target, DNA methyltransferase 1. Ultimately, these events promote cytotoxic T-cell-mediated clearance of tumour cells, which is further enhanced by the addition of immune checkpoint blockade. Our findings indicate that CDK4/6 inhibitors increase tumour immunogenicity and provide a rationale for new combination regimens comprising CDK4/6 inhibitors and immunotherapies as anti-cancer treatment.

The skinny on obesity and cancer.

Obesity now rivals smoking as one of the leading preventable causes of cancer. Obesity-associated neutrophilia is now shown to enhance breast cancer metastasis and to be reversible through dietary modification and weight loss.