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T cells are generally sparse in hormone receptor-positive (HR+) breast cancer, potentially due to limited antigen presentation, but the driving mechanisms of low T cell abundance remains unclear. Therefore, we defined and investigated programs ('gene modules'), related to estrogen receptor signaling (ERS) and immune signaling using bulk and single-cell transcriptome and multiplexed immunofluorescence of breast cancer tissues from multiple clinical sources and human cell lines. The ERS gene module, dominantly expressed in cancer cells, was negatively associated with immune-related gene modules TNFα/NF-κB signaling and type-I interferon (IFN-I) response, which were expressed in distinct stromal and immune cell types, but also, in part, expressed and preserved as a cancer cell-intrinsic mechanisms. Spatial analysis revealed that ERS strongly correlated with reduced T cell infiltration, potentially due to its association with suppression of TNFα/NF-κB-induced angiogenesis and IFN-I-induced HLA expression in macrophages. Preoperative endocrine therapy in ER+/HER2-breast cancer patients produced better responses in ERS-high patients, with TNFα/NF-κB expression associated with reduced ERS. Targeting these pathways may enhance T cell infiltration in HR+ breast cancer patients., Statement of Significance: This study elucidates the immunosuppressive role of ER signaling in breast cancer, highlighting a complex interplay between cancer, stromal, and immune cells and reveals potential approaches to enhance immunogenicity in HR+ breast cancer. These findings offer crucial insights into immune evasion in breast cancer and identify strategies to enhance T cell abundance.

Aging is associated with increased breast cancer risk and outcomes are worse for the oldest and youngest patients, regardless of subtype. It is not known how cells in the breast tumor microenvironment are impacted by age and how they might contribute to age-related disease pathology. Here, we discover age-associated differences in cell states and interactions in human estrogen receptor-positive (ER+) and triple-negative breast cancers (TNBC) using new computational analyses of existing single-cell gene expression data. Age-specific program enrichment (ASPEN) analysis reveals age-related changes, including increased tumor cell epithelial-mesenchymal transition, cancer-associated fibroblast inflammatory responses, and T cell stress responses and apoptosis in TNBC. ER+ breast cancer is dominated by increased cancer cell estrogen receptor 1 ( ESR1) and luminal cell activity, reduced immune cell metabolism, and decreased vascular and extracellular matrix (ECM) remodeling with age. Cell interactome analysis reveals candidate signaling pathways that drive many of these cell states. This work lays a foundation for discovery of age-adapted therapeutic interventions for breast cancer., Competing Interests: Competing interests The authors declare no competing interests.

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., (© 2024 The Authors; Published by the American Association for Cancer Research.)

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., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., Competing Interests: Declaration of interests K. Polyak serves on the scientific advisory boards of Novartis, Vividion Therapeutics, Ideya Biosciences, and Scorpion Therapeutics; holds equity options in Scorpion Therapeutics; has received honorarium from Astra-Zeneca, New Equilibrium Biosciences, and Roche in the past 12 months; and receives sponsored research funding from Novartis. K. Politi is coinventor on a patent for EGFRT790M mutation testing issued, licensed, and with royalties paid from Molecular Diagnostics/Memorial Sloan Kettering Cancer Center. She reports research funding to her institution from AstraZeneca, Roche/Genentech, Boehringer Ingelheim, and D2G Oncology, and consulting for AstraZeneca and Jannssen. K. Palucka is a cofounder of Guardian Bio and holds equity and receives research support from Guardian Bio. She is a member of the scientific advisory board and holds equity from Cue Biopharma. She received research support from Merck in the past. O.A. has received research support from Sanofi and Pacbio in the past. J.J.T. receives patent royalties from Fate Therapeutics. She has also received research support from H3 Biomedicine, Inc. L.M.C. reports consulting services for Cell Signaling Technologies, AbbVie, the Susan G. Komen Foundation, and Shasqi; has received reagent and/or research support from Cell Signaling Technologies, Syndax Pharmaceuticals, ZelBio, Inc., Hibercell, Inc., Acerta Pharma, Prospect Creek Foundation, the Susan G. Komen Foundation, and National Foundation for Cancer Research; and has participated in advisory boards for Syndax Pharmaceuticals, Carisma Therapeutics, Inc., CytomX Therapeutics, Inc., Kineta, Inc., Hibercell, Inc., Cell Signaling Technologies, Alkermes, Inc., Genenta Sciences, Pio Therapeutics, Pty., Ltd., PDX Pharmaceuticals, Inc., NextCure, Guardian Bio, the AstraZeneca Partner of Choice Network, the Lustgarten Foundation, and the NIH/NCI-Frederick National Laboratory Advisory Committee., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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/m 2 for twelve weeks; cohort 2 received weekly paclitaxel 80 mg/m 2 plus every-three-weekly cyclophosphamide 600 mg/m 2 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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

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.

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., (© 2022. The Author(s).)

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.

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., Competing Interests: EM has been compensated for participation on Scientific Advisory Boards for Astra-Zeneca/Medimmune, Celgene, Genentech, Exact Sciences (formerly Genomic Health), Merck, Peregrine Pharmaceuticals, SELLAS Lifescience, and Tapimmune and has had clinical trial support to her former institution (M.D. Anderson Cancer Center) from Astra-Zeneca/Medimmune, EMD-Serono, Galena Biopharma and Genentech, and current institution from Genentech via a SU2C grant. EM has also had sponsored Research Support to the laboratory from GSK and Eli Lilly. JG is a consultant for Glaxo-Smith Kline (GSK), Codagenix, Verseau, Kymera and Array BioPharma and receives sponsored research support from GSK, Array BioPharma and Eli Lilly. ST receives institutional research funding from AstraZeneca, Lilly, Merck, Nektar, Novartis, Pfizer, Genentech/Roche, Immunomedics, Gilead Exelixis, Bristol-Myers Squibb, Eisai, Nanostring, Cyclacel, Odonate, and Seagen; has served as an advisor/consultant to AstraZeneca, Lilly, Merck, Nektar, Novartis, Pfizer, Genentech/Roche, Immunomedics, Bristol-Myers Squibb, Eisai, Nanostring, Seagen, Puma, Sanofi, Celldex, Paxman, Puma, Silverback Therapeutics, G1 Therapeutics, AbbVie, Anthenex, OncoPep, Outcomes4Me, Kyowa Kirin Pharmaceuticals, Daiichi-Sankyo, Gilead and Samsung Bioepsis Inc. AW receives institutional research funding from Genentech/Roche. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Goldberg, Pastorello, Vallius, Davis, Cui, Agudo, Waks, Keenan, McAllister, Tolaney, Mittendorf and Guerriero.)

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 ., (©2019 American Association for Cancer Research.)

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., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2018. Published by Elsevier Ltd.)

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 ., (©2018 American Association for Cancer Research.)

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-1β (IL-1β)-expressing innate immune cells that infiltrate distant MIC microenvironments. At the metastatic site, IL-1β 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-1β 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.

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., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

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., Competing Interests: The authors have declared that no competing interests exist.

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.

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.

Background: The bone-targeting agent zoledronic acid (ZOL) increases breast cancer survival in subsets of patients, but the underlying reasons for this protective effect are unknown. ZOL modulates the activity of osteoclasts and osteoblasts, which form hematopoietic stem cell niches, and therefore may affect hematopoietic cells that play a role in breast cancer progression., Method: Immunocompetent and immunocompromised strains of mice commonly used for breast cancer research were injected with a single, clinically relevant dose of ZOL (100 μg/kg) or vehicle control. The effects of ZOL on the bone marrow microenvironment (bone volume, bone cell number/activity, extracellular matrix composition) were established at various time points following treatment, using micro-computed tomography (μCT) analysis, histomorphometry, ELISA and immunofluorescence. The effects on peripheral blood and bone marrow hematopoietic progenitor populations were assessed using a HEMAVET® hematology analyzer and multicolor flow cytometry, respectively. Tumor support function of bone marrow cells was determined using an in vivo functional assay developed in our laboratory., Results: Using multiple mouse strains, we observed transient changes in numbers of hematopoietic stem cells, myeloid-biased progenitor cells, and lymphoid-biased cells concurrent with changes to hematopoietic stem cell niches following ZOL administration. Importantly, bone marrow cells from mice treated with a single, clinically relevant dose of ZOL inhibited breast tumor outgrowth in vivo. The ZOL-induced tumor suppressive function of the bone marrow persisted beyond the time point at which numbers of hematopoietic progenitor cells had returned to baseline., Conclusions: These findings provide novel evidence that alterations to the bone marrow play a role in the anti-tumor activity of ZOL and suggest possibilities for capitalizing on the beneficial effects of ZOL in reducing breast cancer development and progression.

Luminal breast cancers are typically estrogen receptor-positive and generally have the best prognosis. However, a subset of luminal tumors, namely luminal B cancers, frequently metastasize and recur. Unfortunately, the causal events that drive their progression are unknown, and therefore it is difficult to identify individuals who are likely to relapse and should receive escalated treatment. Here, we identify a bifunctional RasGAP tumor suppressor whose expression is lost in almost 50% of luminal B tumors. Moreover, we show that two RasGAP genes are concomitantly suppressed in the most aggressive luminal malignancies. Importantly, these genes cooperatively regulate two major oncogenic pathways, RAS and NF-κB, through distinct domains, and when inactivated drive the metastasis of luminal tumors in vivo Finally, although the cooperative effects on RAS drive invasion, NF-κB activation triggers epithelial-to-mesenchymal transition and is required for metastasis. Collectively, these studies reveal important mechanistic insight into the pathogenesis of luminal B tumors and provide functionally relevant prognostic biomarkers that may guide treatment decisions., Significance: The lack of insight into mechanisms that underlie the aggressive behavior of luminal B breast cancers impairs treatment decisions and therapeutic advances. Here, we show that two RasGAP tumor suppressors are concomitantly suppressed in aggressive luminal B tumors and demonstrate that they drive metastasis by activating RAS and NF-κB. Cancer Discov; 7(2); 202-17. ©2016 AACR.See related commentary by Sears and Gray, p. 131This article is highlighted in the In This Issue feature, p. 115., (©2016 American Association for Cancer Research.)

Despite the high incidence of oncogenic mutations in PIK3CA, the gene encoding the catalytic subunit of PI3K, PI3K inhibitors have yielded little clinical benefit for breast cancer patients. Recent epidemiologic studies have suggested a therapeutic benefit from aspirin intake in cancers harboring oncogenic PIK3CA Here, we show that mutant PIK3CA-expressing breast cancer cells have greater sensitivity to aspirin-mediated growth suppression than their wild-type counterparts. Aspirin decreased viability and anchorage-independent growth of mutant PIK3CA breast cancer cells independently of its effects on COX-2 and NF-κB. We ascribed the effects of aspirin to AMP-activated protein kinase (AMPK) activation, mTORC1 inhibition, and autophagy induction. In vivo, oncogenic PIK3CA-driven mouse mammary tumors treated daily with aspirin resulted in decreased tumor growth kinetics, whereas combination therapy of aspirin and a PI3K inhibitor further attenuated tumor growth. Our study supports the evaluation of aspirin and PI3K pathway inhibitors as a combination therapy for targeting breast cancer. Cancer Res; 77(3); 790-801. ©2016 AACR., Competing Interests: The authors disclose no potential conflict of interest, (©2016 American Association for Cancer Research.)

Systemic factors including cytokines, cell-free nucleic acids, microvesicles, and platelets are appreciated as important regulators of adenocarcinoma progression. Research findings using pre-clinical mouse models have revealed that many such systemically acting factors are either secreted by or responsive to peripheral tumors and impact bone and bone marrow (collectively referred to as the bone microenvironment) to initiate processes that ultimately govern disease progression, even in the absence of detectable bone metastases. In some cases, cancer-driven modulation of the bone microenvironment involves mobilization of bone marrow hematopoietic and mesenchymal cells into the circulation that are subsequently recruited into peripheral tissues and tumors. In other cases, systemic factors alter bone marrow cell (BMC) differentiation and/or gene expression to render the BMCs pro-tumorigenic even prior to their mobilization into the circulation. Given their effect on the bone microenvironment, it stands to reason that such systemic factors might also influence metastases in the bone; however, this hypothesis remains to be comprehensively tested. Here, we briefly review what is known, and not known, about systemic factors that regulate the bone microenvironment and thereby influence bone metastases. We also pose a number of currently unanswered questions in this active area of research. A better understanding of systemic processes that influence bone metastasis should aid discovery of therapeutic approaches that aim to eradicate or reduce disease burden in the bone, which is the cause of significant patient mortality and morbidity and is currently incurable.

Triple-negative breast cancer (TNBC) is considered an early onset subtype of breast cancer that carries with it a poorer prognosis in young rather than older women for reasons that remain poorly understood. Hematopoiesis in the bone marrow becomes altered with age and may therefore affect the composition of tumor-infiltrating hematopoietic cells and subsequent tumor progression. In this study, we investigated how age- and tumor-dependent changes to bone marrow-derived hematopoietic cells impact TNBC progression. Using multiple mouse models of TNBC tumorigenesis and metastasis, we found that a specific population of bone marrow cells (BMC) upregulated CSF-1R and secreted the growth factor granulin to support stromal activation and robust tumor growth in young mice. However, the same cell population in old mice expressed low levels of CSF1R and granulin and failed to promote tumor outgrowth, suggesting that age influences the tumorigenic capacity of BMCs in response to tumor-associated signals. Importantly, BMCs from young mice were sufficient to activate a tumor-supportive microenvironment and induce tumor progression in old mice. These results indicate that hematopoietic age is an important determinant of TNBC aggressiveness and provide rationale for investigating age-stratified therapies designed to prevent the protumorigenic effects of activated BMCs. Cancer Res; 76(10); 2932-43. ©2016 AACR., (©2016 American Association for Cancer Research.)

Large wildfires of increasing frequency and severity threaten local populations and natural resources and contribute carbon emissions into the earth-climate system. Although wildfires have been researched and modeled for decades, no verifiable physical theory of spread is available to form the basis for the precise predictions needed to manage fires more effectively and reduce their environmental, economic, ecological, and climate impacts. Here, we report new experiments conducted at multiple scales that appear to reveal how wildfire spread derives from the tight coupling between flame dynamics induced by buoyancy and fine-particle response to convection. Convective cooling of the fine-sized fuel particles in wildland vegetation is observed to efficiently offset heating by thermal radiation until convective heating by contact with flames and hot gasses occurs. The structure and intermittency of flames that ignite fuel particles were found to correlate with instabilities induced by the strong buoyancy of the flame zone itself. Discovery that ignition in wildfires is critically dependent on nonsteady flame convection governed by buoyant and inertial interaction advances both theory and the physical basis for practical modeling.

Recent pre-clinical and clinical research has provided evidence that cancer progression is driven not only by a tumour's underlying genetic alterations and paracrine interactions within the tumour microenvironment, but also by complex systemic processes. We review these emerging paradigms of cancer pathophysiology and discuss how a clearer understanding of systemic regulation of cancer progression could guide development of new therapeutic modalities and efforts to prevent disease relapse following initial diagnosis and treatment.

Summary: Disease recurrence is the most common cause of death for patients with breast cancer, yet little is known about the molecular mechanisms underlying this process. Using inducible transgenic mouse model systems, Feng and colleagues identified SPSB1 as a determinant of breast cancer recurrence by virtue of its ability to protect tumor cells from apoptosis through c-MET activation., (©2014 American Association for Cancer Research.)

Unlabelled: Neoplastic cells rely on the tumor microenvironment (TME) for survival and progression factors. Indeed, senescent and cancer-associated fibroblasts (CAF) express factors that promote tumorigenesis that are collectively referred to as the senescence-associated secretory phenotype (SASP). Despite their importance in tumorigenesis, the mechanisms that control TME-derived factor expression remain poorly understood. Here, we address a key unanswered question: how the SASP is sustained in senescent fibroblasts and CAFs. We find that the mitogen-activated protein kinase p38 (p38MAPK) controls AUF1 occupancy on SASP mRNAs and thus controls their stability. The importance of this regulatory mechanism is underscored by our findings that stromal-specific p38MAPK inhibition abrogates the tumor-promoting activities of CAFs and senescent fibroblasts. Our data suggest that targeting SASP mRNA stability through inhibition of p38MAPK will significantly aid the development of clinical strategies to target the TME., Significance: The TME plays a key role in tumorigenesis. We demonstrate that p38MAPK governs a posttranscriptional mechanism that sustains the protumorigenic SASP. Inhibition of p38MAPK abrogates the tumor-promoting activities of CAFs and senescent fibroblasts. Thus, p38MAPK is a TME-specific Achilles' heel that may be exploited as a new therapeutic target., (©2014 American Association for Cancer Research.)

The causes for malignant progression of disseminated tumors and the reasons recurrence rates differ in women with different breast cancer subtypes are unknown. Here, we report novel mechanisms of tumor plasticity that are mandated by microenvironmental factors and show that recurrence rates are not strictly due to cell-intrinsic properties. Specifically, outgrowth of the same population of incipient tumors is accelerated in mice with triple-negative breast cancer (TNBC) relative to those with luminal breast cancer. Systemic signals provided by overt TNBCs cause the formation of a tumor-supportive microenvironment enriched for EGF and insulin-like growth factor-I (IGF-I) at distant indolent tumor sites. Bioavailability of EGF and IGF-I enhances the expression of transcription factors associated with pluripotency, proliferation, and epithelial-mesenchymal transition. Combinatorial therapy with EGF receptor and IGF-I receptor inhibitors prevents malignant progression. These results suggest that plasticity and recurrence rates can be dictated by host systemic factors and offer novel therapeutic potential for patients with TNBC.

Breast cancer is now the most frequently diagnosed cancer and leading cause of cancer death in women worldwide. Strategies targeting the primary tumour have markedly improved, but systemic treatments to prevent metastasis are less effective; metastatic disease remains the underlying cause of death in the majority of patients with breast cancer who succumb to their disease. The long latency period between initial treatment and eventual recurrence in some patients suggests that a tumour may both alter and respond to the host systemic environment to facilitate and sustain disease progression. Results from studies in animal models suggest that specific subtypes of breast cancer may direct metastasis through recruitment and activation of haematopoietic cells. In this review, we focus on data implicating breast cancer as a systemic disease., (© 2013 The Association for the Publication of the Journal of Internal Medicine.)

Studies of epithelial cancers (i.e., carcinomas) traditionally focused on transformation of the epithelium (i.e., the cancer cells) and how aberrant signaling within the cancer cells modulates the surrounding tissue of origin. In more recent decades, the normal cells, blood vessels, molecules, and extracellular components that surround the tumor cells, collectively known as the "tumor microenvironment" or "stroma", have received increasing attention and are now thought to be key regulators of tumor initiation and progression. Of particular relevance to the work reviewed herein are the fibroblasts, which make up the major cell type within the microenvironment of most carcinomas. Due to their inherent heterogeneity, plasticity, and function, it is perhaps not surprising that fibroblasts are ideal modulators of normal and cancerous epithelium; however, these aspects also present challenges if we are to interrupt their tumor-supportive functions. Here, we review the current body of knowledge and the many questions that still remain about the special entity known as the cancer-associated fibroblast. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease., (Copyright © 2012. Published by Elsevier B.V.)

Unlabelled: Breast cancer recurrence rates vary following treatment, suggesting that tumor cells disseminate early from primary sites but remain indolent indefinitely before progressing to symptomatic disease. The reasons why some indolent disseminated tumors erupt into overt disease are unknown. We discovered a novel process by which certain luminal breast cancer (LBC) cells and patient tumor specimens (LBC "instigators") establish a systemic macroenvironment that supports outgrowth of otherwise-indolent disseminated tumors ("responders"). Instigating LBCs secrete cytokines that are absorbed by platelets, which are recruited to responding tumor sites where they aid vessel formation. Instigator-activated bone marrow cells enrich responding tumor cell expression of CD24, an adhesion molecule for platelets, and provide a source of VEGF receptor 2(+) tumor vessel cells. This cascade results in growth of responder adenocarcinomas and is abolished when platelet activation is inhibited by aspirin. These findings highlight the macroenvironment as an important component of disease progression that can be exploited therapeutically., Significance: Currently, processes that mediate progression of otherwise indolent tumors are not well understood, making it difficult to accurately predict which cancer patients are likely to relapse. Our findings highlight the macroenvironment as an important component of disease progression that can be exploited to more accurately identify patients who would benefit from adjuvant therapy., (©2012 AACR.)

Tumors have been increasingly recognized as organs with a complexity that approaches, and may even exceed, that of healthy tissues. When viewed from this perspective, the biology of a tumor can be understood only by studying tumor cell heterogeneity and the microenvironment that is constructed during the course of tumorigenesis and malignant progression. Recent work has revealed the existence of cancer stem cells, the "bugs", with the capacity for self-renewal and tumor propagation. In addition, it is now recognized that the tumor microenvironment, the "bed", plays a critical role in supporting cancer stem cells and also may promote neoplasia and malignant progression. The interdependence of the cell-intrinsic features of cancer, including the cancer stem cell "bugs" and the tumor microenvironment "bed", is only beginning to be understood. In this review, we highlight the rapidly evolving concepts about the interactions between tumor stem cells and their microenvironment, the insights gained from studying their normal tissue counterparts, and the questions and controversies surrounding this area of research, with an emphasis on breast and lung cancer. Finally, we address evidence supporting the notion that eliminating the bed as well as the bugs should lead to more effective and personalized cancer treatments that improve patient outcome., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

New mouse models shed light on mechanisms of lung cancer progression and pinpoint Rac1b as a potential therapeutic target (Stallings-Mann et al., this issue).

Systemic instigation is a process by which endocrine signals sent from certain tumors (instigators) stimulate BM cells (BMCs), which are mobilized into the circulation and subsequently foster the growth of otherwise indolent carcinoma cells (responders) residing at distant anatomical sites. The identity of the BMCs and their specific contribution or contributions to responder tumor growth have been elusive. Here, we have demonstrated that Sca1+ cKit- hematopoietic BMCs of mouse hosts bearing instigating tumors promote the growth of responding tumors that form with a myofibroblast-rich, desmoplastic stroma. Such stroma is almost always observed in malignant human adenocarcinomas and is an indicator of poor prognosis. We then identified granulin (GRN) as the most upregulated gene in instigating Sca1+ cKit- BMCs relative to counterpart control cells. The GRN+ BMCs that were recruited to the responding tumors induced resident tissue fibroblasts to express genes that promoted malignant tumor progression; indeed, treatment with recombinant GRN alone was sufficient to promote desmoplastic responding tumor growth. Further, analysis of tumor tissues from a cohort of breast cancer patients revealed that high GRN expression correlated with the most aggressive triple-negative, basal-like tumor subtype and reduced patient survival. Our data suggest that GRN and the unique hematopoietic BMCs that produce it might serve as novel therapeutic targets.

Breast cancer is the most common malignancy among women worldwide and is the most common cause of death for women between 35 and 50 years of age. Women with breast cancer are at risk of developing metastases for their entire lifetime and, despite local and systemic therapies, approximately 30% of breast cancer patients will relapse (Jemal et al., 2010). Nearly all breast cancer related deaths are due to metastatic disease, even though metastasis is considered to be an inefficient process. In some cases, tumor cells disseminate from primary sites at an early stage, but remain indolent for protracted periods of time before becoming overt, life-threatening tumors. Little is known about the mechanisms that cause these indolent tumors to grow into malignant disease. Because of this gap in our understanding, we are unable to predict which breast cancer patients are likely to experience disease relapse or develop metastases years after treatment of their primary tumor. A better understanding of the mechanisms and signals involved in the exit of tumor cells from dormancy would not only allow for more accurate selection of patients that would benefit from systemic therapy, but could also lead to the development of more targeted therapies to inhibit the signals that promote disease progression. In this review, we address the systemic, or "macroenvironmental", contribution to tumor initiation and progression and what is known about how a pro-tumorigenic systemic environment is established.

Carcinomas are composed of neoplastic epithelial cells, which form the heart of the tumor, as well as a variety of mesenchymal cell types and extracellular matrix components that comprise the tumor stroma, often termed its microenvironment. The normal counterparts of some stromal cells are thought to limit tumor growth, while tumor-associated stromal cells have been convincingly shown to actively promote tumor progression via complex heterotypic interactions with the nearby carcinoma cells. More recent advances have revealed that tumor-host interactions extend well beyond the local tissue microenvironment (ie, interactions between the neoplastic cells and the nearby stroma) and that tumors not only respond to, but actively perturb host organs at distant anatomic sites. This indicates that many aspects of tumor biology can only be explained by a detailed understanding of both local and systemic interactions, yet we currently have only a fragmentary understanding of both processes. In this review, we address the recent advances in our understanding of the contributions of local and systemic environments to cancer progression, the ability of tumors to actively perturb the host environment, and current therapeutic approaches that are designed to disrupt tumor-host relationships.

The p53 tumor suppressor is a key mediator of cellular responses to various stresses. Here, we show that under conditions of basal physiologic and cell-culture stress, p53 inhibits expression of the CD44 cell-surface molecule via binding to a noncanonical p53-binding sequence in the CD44 promoter. This interaction enables an untransformed cell to respond to stress-induced, p53-dependent cytostatic and apoptotic signals that would otherwise be blocked by the actions of CD44. In the absence of p53 function, the resulting derepressed CD44 expression is essential for the growth and tumor-initiating ability of highly tumorigenic mammary epithelial cells. In both tumorigenic and nontumorigenic cells, CD44's expression is positively regulated by p63, a paralogue of p53. Our data indicate that CD44 is a key tumor-promoting agent in transformed tumor cells lacking p53 function. They also suggest that the derepression of CD44 resulting from inactivation of p53 can potentially aid the survival of immortalized, premalignant cells.

The effects of primary tumors on the host systemic environment and resulting contributions of the host to tumor growth are poorly understood. Here, we find that human breast carcinomas instigate the growth of otherwise-indolent tumor cells, micrometastases, and human tumor surgical specimens located at distant anatomical sites. This systemic instigation is accompanied by incorporation of bone-marrow cells (BMCs) into the stroma of the distant, once-indolent tumors. We find that BMCs of hosts bearing instigating tumors are functionally activated prior to their mobilization; hence, when coinjected with indolent cells, these activated BMCs mimic the systemic effects imparted by instigating tumors. Secretion of osteopontin by instigating tumors is necessary for BMC activation and the subsequent outgrowth of the distant otherwise-indolent tumors. These results reveal that outgrowth of indolent tumors can be governed on a systemic level by endocrine factors released by certain instigating tumors, and hold important experimental and therapeutic implications.

Hepatocyte growth factor (HGF) signaling via its receptor, the proto-oncogene Met, alters cell proliferation and motility and has been associated with tumor metastasis. HGF treatment of HepG2 human hepatocellular carcinoma cells induces cell migration concomitant with increased levels of the p27(kip1) cyclin-cdk inhibitor. HGF signaling resulted in nuclear export of endogenous p27 to the cytoplasm, via Ser-10 phosphorylation, where it colocalized with F-actin. Introduction of transducible p27 protein (TATp27) was sufficient for actin cytoskeletal rearrangement and migration of HepG2 cells. TATp27 mutational analysis identified a novel p27 C-terminal domain required for cell migration, distinct from the N-terminal cyclin-cyclin-dependent kinase (cdk) binding domain. Loss or disruption of the p27 C-terminal domain abolished both actin rearrangement and cell migration. The cell-scattering activity of p27 occurred independently of its cell cycle arrest functions and required cytoplasmic localization of p27 via Ser-10 phosphorylation. Furthermore, Rac GTPase was necessary for p27-dependent migration but alone was insufficient for HepG2 cell migration. These results predicted a migration defect in p27-deficient cells. Indeed, p27-deficient primary fibroblasts failed to migrate, and reconstitution with TATp27 rescued the motility defect. These observations define a novel role for p27 in cell motility that is independent of its function in cell cycle inhibition.

Manipulation of mammalian cells has been achieved by the transfection of expression vectors, microinjection, or diffusion of peptidyl mimetics. While these approaches have been somewhat successful, the classic manipulation methods are not easily regulated and can be laborious. One approach to circumvent these problems is the use of HIV TAT-mediated protein transduction. Although this technology was originally described in 1988, few improvements were reported in the subsequent 10 years. In the last few years, significant steps have been taken to advance this technology into a broadly applicable method that allows for the rapid introduction of full-length proteins into primary and transformed cells. The technology requires the synthesis of a fusion protein, linking the TAT transduction domain to the molecule of interest using a bacterial expression vector, followed by the purification of this fusion protein under either soluble or denaturing conditions. The purified fusion protein can be directly added to mammalian cell culture or injected in vivo into mice. Protein transduction occurs in a concentration-dependent manner, achieving maximum intracellular concentrations in less than 5 min, with nearly equal intracellular concentrations between all cells in the transduced population. Full-length TAT fusion proteins have been used to address a number of biological questions, relating to cell cycle progression, apoptosis, and cellular architecture. Described here are the fundamental requirements for the creation, isolation, and utilization of TAT-fusion proteins to affect mammalian cells. A detailed protocol for production and transduction of TAT-Cdc42 into primary cells is given to illustrate the technique., (Copyright 2001 Academic Press.)