• Animals
• Apoptosis
• Arginase/genetics
• Arginase/metabolism
• Cell Proliferation
• Cell Transformation, Neoplastic/genetics
• Cell Transformation, Neoplastic/immunology
• Cell Transformation, Neoplastic/metabolism
• Cell Transformation, Neoplastic/pathology
• Female
• Humans
• Mammary Neoplasms, Experimental/genetics
• Mammary Neoplasms, Experimental/immunology
• Mammary Neoplasms, Experimental/metabolism
• Mammary Neoplasms, Experimental/pathology
• Mice
• Mice, Transgenic
• Tumor Cells, Cultured
• Tumor Microenvironment/immunology

• Biomarker
• DCIS
• Immune
• Prognostic
• Progression
• Recurrence

One third of newly diagnosed breast cancers in the US are early-stage lesions. The etiological understanding and treatment of these lesions have become major clinical challenges. Because breast cancer risk factors are often linked to aberrant nitric oxide (NO) production, we hypothesized that abnormal NO levels might contribute to the formation of early-stage breast lesions. We recently reported that the basal level of NO in the normal breast epithelia plays crucial roles in tissue homeostasis, whereas its reduction contributes to the malignant phenotype of cancer cells. Here, we show that the basal level of NO in breast cells plummets during cancer progression due to reduction of the NO synthase cofactor, BH₄, under oxidative stress. Importantly, pharmacological deprivation of NO in prepubertal to pubertal animals stiffens the extracellular matrix and induces precancerous lesions in the mammary tissues. These lesions overexpress a fibrogenic cytokine, TGFβ, and an oncogene, ERBB2, accompanied by the occurrence of senescence and stem cell-like phenotype. Consistently, normalization of NO levels in precancerous and cancerous breast cells downmodulates TGFβ and ERBB2 and ameliorates their proliferative phenotype. This study sheds new light on the etiological basis of precancerous breast lesions and their potential prevention by manipulating the basal NO level.

• biological network
• carcinogenesis
• colorectal cancer
• prognostic genes
• random walk

Studies assessing immune parameters typically utilize human PBMCs or murine splenocytes to generate data that is interpreted as representative of immune status. Using splenocytes, we have shown memory CD4-T cells that expand following systemic immunostimulatory therapies undergo rapid IFNg-mediated activation induced cell death (AICD) resulting in a net loss of total CD4-T cells which correlates with elevated PD-1 expression. This is in contrast to CD8-T cells which expand with minimal PD-1 upregulation and apoptosis. In this study we expand upon our previous work by evaluating CD4 and CD8-T cell phenotype and distribution in peripheral organs which are more representative of immune responses occurring at metastatic sites following immunotherapy.

Phenotypic assessment of T cells in both lymphoid (spleen and LN) as well as peripheral organs (liver and lungs) in control and immunotherapy treated mice was performed to survey the impact of location on memory phenotype and activation marker status. Peripheral blood from patients undergoing systemic high dose IL-2 was also assessed for expression of PD-1 and memory phenotype.

Here we reveal that, similar to what occurs in the spleen and lymph nodes, CD4-T cell numbers decreased while CD8-T cells expanded at these peripheral sites. In contrast to having differential expression of PD-1 as occurs in the spleen, both CD4 and CD8-T cells had significantly elevated levels of PD-1 in both the liver and lungs. Further analysis correlated PD-1 expression to CD62L^low (T effector/effector memory,T_E/EM) expression which are more prevalent in CD4-T cells in general as well as CD8-T cells in peripheral organs. Similar elevated PD-1 expression on T_E/EM cells was observed in patients undergoing systemic high-dose IL-2 therapy.

These data highlight PD-1 expressing and/or T_E/EM subsets of T cells in circulation as more representative of cells at immune sites and underscore the importance of valuation both in lymphoid as well as target organs when making determinations about immune status.

ClinicalTrials.gov NCT01416831. Registered August 12, 2011.

The online version of this article (doi:10.1186/s40425-017-0235-4) contains supplementary material, which is available to authorized users.

• Bystander Activation
• CD8
• Cancer
• Immunotherapy
• NKG2D
• PD-1

• BREAST CANCER
• BREAST PATHOLOGY
• TUMOUR IMMUNITY
• TUMOUR MARKERS

• ductal carcinoma in situ
• glucose uptake
• intracellular pH
• mouse mammary carcinoma model
• proton export
• tumor heterogeneity
• tumor microenvironment

Ductal carcinoma in situ (DCIS) is a category of early stage, non-invasive breast tumor defined by the intraductal proliferation of malignant breast epithelial cells. DCIS is a heterogeneous disease composed of multiple molecular subtypes including luminal, HER2 and basal-like types, which are characterized by immunohistochemical analyses and gene expression profiling. Following surgical and radiation therapies, patients with luminal-type, estrogen receptor-positive DCIS breast tumors can benefit from adjuvant endocrine-based treatment. However, there are no available targeted therapies for patients with basal-like DCIS (BL-DCIS) tumors due to their frequent lack of endocrine receptors and HER2 amplification, rendering them potentially susceptible to recurrence. Moreover, multiple lines of evidence suggest that DCIS is a non-obligate precursor of invasive breast carcinoma. This raises the possibility that targeting precursor BL-DCIS is a promising strategy to prevent BL-DCIS patients from the development of invasive basal-like breast cancer. An accumulating body of evidence demonstrates the existence of cancer stem-like cells (CSCs) in BL-DCIS, which potentially determine the features of BL-DCIS and their ability to progress into invasive cancer. This review encompasses the current knowledge in regard to the characteristics of BL-DCIS, identification of CSCs, and their biological properties in BL-DCIS. We summarize recently discovered relevant molecular signaling alterations that promote the generation of CSCs in BL-DCIS and the progression of BL-DCIS to invasive breast cancer, as well as the influence of the tissue microenvironment on CSCs and the invasive transition. Finally, we discuss the translational implications of these findings for the prognosis and prevention of BL-DCIS relapse and progression.

• Ductal carcinoma in situ
• Invasive ductal carcinoma
• Basal-like ductal carcinoma in situ
• Basal-like invasive ductal carcinoma
• Cancer stem cells

• R01 CA157779 NCI NIH HHS
• R01 CA163820 NCI NIH HHS

• greedy searching
• lung squamous carcinoma
• multi-stage carcinogenesis
• network-based
• prognostic module

• Adult
• Aged
• Carcinoma, Squamous Cell/genetics
• Carcinoma, Squamous Cell/pathology
• Female
• Gene Expression Profiling
• Gene Regulatory Networks
• Humans
• Lung Neoplasms/genetics
• Lung Neoplasms/pathology
• Male
• Middle Aged
• Prognosis

• Animals
• Autoradiography
• Doxorubicin/administration & dosage
• Doxorubicin/pharmacokinetics
• Female
• High-Intensity Focused Ultrasound Ablation
• Liposomes/pharmacokinetics
• Magnetic Resonance Imaging
• Mammary Neoplasms, Experimental/metabolism
• Mammary Neoplasms, Experimental/mortality
• Mammary Neoplasms, Experimental/therapy
• Mice
• Mice, Inbred BALB C
• Positron-Emission Tomography
• Tissue Distribution

• R01CA134659 NCI NIH HHS
• R01 CA103828 NCI NIH HHS
• R01CA199658 NCI NIH HHS
• R01CA103828 NCI NIH HHS
• R01 CA134659 NCI NIH HHS
• R01 CA112356 NCI NIH HHS
• R01 CA199658 NCI NIH HHS

The diversity of human breast cancer subtypes has led to the hypothesis that breast cancer is actually a number of different diseases arising from cells at various stages of differentiation. The elusive nature of the cell(s) of origin thus hampers approaches to eradicate the disease.

Clonal cell lines were isolated from primary transgenic polyomavirus middle T (PyVmT) luminal tumors. Mammary cancer stem cell (MaCSC) properties were examined by immunofluorescence, flow cytometry, differentiation assays and in vivo tumorigenesis.

Clonal cell lines isolated from primary PyVmT mouse mammary luminal tumors can differentiate into luminal, myoepithelial, alveolar and adipocyte lineages. Upon orthotopic injection, progeny of a single cell follow a pattern of progression from ductal carcinoma in situ, to adenoma, adenocarcinoma and epithelial metastasis that recapitulates the transgenic model. Tumors can evolve in vivo from hormone receptor-positive to hormone receptor-negative Her2-positive, or triple negative CD44hi basal-like and claudin-low tumors. Contrary to the current paradigm, we have defined a model in which multiple tumor subtypes can originate from a single multipotent cancer stem cell that undergoes genetic and/or epigenetic evolution during tumor progression. As in human tumors, the more aggressive tumor subtypes express nuclear p53. Tumor cell lines can also be derived from these more advanced tumor subtypes.

Since the majority of human tumors are of the luminal subtype, understanding the cell of origin of these tumors and how they relate to other tumor subtypes will impact cancer therapy. Analysis of clonal cell lines derived from different tumor subtypes suggests a developmental hierarchy of MaCSCs, which may provide insights into the progression of human breast cancer.

The online version of this article (doi:10.1186/s13058-015-0615-y) contains supplementary material, which is available to authorized users.

• mammary duct
• breast cancer
• ductal carcinoma in situ
• microfluidics
• 3d
• lumen
• microenvironment
• extracellular matrix

• Breast Neoplasms/pathology
• Carcinoma, Intraductal, Noninfiltrating/pathology
• Cell Line, Tumor
• Coculture Techniques
• Female
• Humans
• In Vitro Techniques/instrumentation
• In Vitro Techniques/methods
• Microfluidics
• Models, Biological
• Neoplasm Invasiveness

• P30 CA014520 NCI NIH HHS
• R01 EB010039 NIBIB NIH HHS
• T32 HL007899 NHLBI NIH HHS
• P30CA014520 NCI NIH HHS

• Breast cancer
• Ductal carcinoma in situ (DCIS)
• cancer progression
• epithelial cells
• invasive breast carcinoma (IBC)
• microenvironment

• Breast Neoplasms/genetics
• Breast Neoplasms/metabolism
• Breast Neoplasms/pathology
• Breast Neoplasms/therapy
• Carcinoma, Intraductal, Noninfiltrating/genetics
• Carcinoma, Intraductal, Noninfiltrating/metabolism
• Carcinoma, Intraductal, Noninfiltrating/pathology
• Carcinoma, Intraductal, Noninfiltrating/therapy
• Female
• Humans

As the field of molecular imaging evolves and increasingly is asked to fill the discovery and validation space between basic science and clinical applications, careful consideration should be given to the models in which studies are conducted. The MIN-O mouse model series is an established in vivo model of human mammary precancer ductal carcinoma in situ with progression to invasive carcinoma. This series of transplant lines is propagated in vivo and experiments utilizing this model can be completed in non-engineered immune intact FVB/n wild type mice thereby modeling the tumor microenvironment with biological relevance superior to traditional tumor cell xenografts. Unfortunately, the same qualities that make this and many other transplant lines more biologically relevant than standard cell lines for molecular imaging studies present a significant obstacle as somatic genetic re-engineering modifications common to many imaging applications can be technically challenging. Here, we describe a protocol for the efficient lentiviral transduction of cell slurries derived from precancerous MIN-O lesions, in vitro culture of “MIN-O-spheres” derived from single cell clones, and the subsequent transplantation of these spheres to produce transduced sublines suitable for optical imaging applications. These lines retain the physiologic and pathologic properties, including multilineage differentiation, and complex microanatomic interaction with the host stroma characteristic of the MIN-O model. We also present the in vivo imaging and immunohistochemical analysis of serial transplantation of one such subline and detail the progressive multifocal loss of the transgene in successive generations.

• Animals
• Breast Neoplasms
• Cell Line
• Cell Line, Tumor
• Disease Models, Animal
• Humans
• Immunohistochemistry
• Lentivirus/genetics
• Mice
• Molecular Imaging/methods
• Neoplasm Transplantation
• Polymerase Chain Reaction

• P30 CA093373 NCI NIH HHS
• U01 CA141582 NCI NIH HHS
• K26 RR024037 NCRR NIH HHS
• RC4 EB012836 NIBIB NIH HHS
• U01 CA14582 NCI NIH HHS

It is still uncertain whether metastasis is predominantly an early or late event in tumor progression. The detection of early metastases and cells responsible for the dissemination may therefore have significant clinical implications.

Lung dissemination and/or metastasis were investigated in mice carrying the polyomavirus middle-T oncogene (PyMT) during different stages of mammary tumorigenesis using the colony forming assay. Immunocytochemical or immunohistochemical staining was used to identify subpopulations of cells responsible for lung dissemination and metastasis. Histological examination was used to show primary and metastatic tumors. The tumor-initiating and metastatic capacity of cells expressing stem cell markers was assessed in syngeneic wild-type (WT) mice whose mammary fat pads were injected with these cells.

Metastatic mammary epithelial cells were detected in the lungs of mice carrying the PyMT oncogene (MMT mice). These cells were observed early in breast tumorigenesis when the mammary tree appeared by histological inspection to be normal (or at a premalignant stage), suggesting the possession of disseminating and metastatic capacity even before full malignant transformation. Some of the disseminated cells and lung metastases displayed surface stem cell markers. These findings suggest that stem cells from apparently precancerous primary lesions could be a source of metastasis. Indeed, injection of lung tissue cells from MMT mice into syngeneic WT mice resulted in the formation of mammary tumors. These tumors resembled their parent mammary tumors in the MMT donors as well as grafted tumors derived from mammary tumor cells. Furthermore, when we injected lung tissue cells from GFP MMT mice into the fat pads of recipient WT mice, disseminated or metastatic GFP-expressing cells were detected in the lungs, lymph nodes and blood of the recipient WT mice. We finally identified a subpopulation of mammary epithelial/tumor cells expressing CD44 and Sca1 that was largely responsible for dissemination and metastasis in MMT mice.

The tumorigenic and metastatic potential of a subpopulation of mammary epithelial/tumor cells in MMT mice is endowed relatively early in mammary neoplasms and suggests a potential role for cancer stem cell sub-populations in metastasis.

• Antigens, CD
• Biomedical Research/trends
• Breast Neoplasms/genetics
• Breast Neoplasms/pathology
• Cadherins/genetics
• Epithelial-Mesenchymal Transition/genetics
• Female
• Gene Expression Regulation, Neoplastic
• Humans
• Neoplasm, Residual/pathology
• Neoplastic Stem Cells/pathology
• Phenotype

• P30 CA093373 NCI NIH HHS
• U01 CA141582 NCI NIH HHS

• cancer stem cells
• infection
• cancer etiology
• cancer signaling

• Animals
• Bacterial Infections/complications
• Bacterial Infections/pathology
• Cell Proliferation
• DNA Methylation
• Female
• Humans
• Male
• Mice
• Mutation
• Neoplasms/etiology
• Neoplasms/genetics
• Neoplasms/pathology
• Neoplastic Stem Cells/pathology
• Signal Transduction
• Stem Cells/microbiology
• Stem Cells/parasitology
• Stem Cells/pathology
• Stem Cells/physiology
• Trematode Infections/complications
• Trematode Infections/pathology
• Virus Diseases/complications
• Virus Diseases/pathology
• Viruses/genetics
• Viruses/metabolism

• R01 CA112481 NCI NIH HHS

• Animals
• Breast Neoplasms/pathology
• Carcinoma, Ductal, Breast/pathology
• Carcinoma, Intraductal, Noninfiltrating/pathology
• Disease Progression
• Female
• Humans
• Mice

• R21 CA125698 NCI NIH HHS

• Animals
• Argonaute Proteins
• Base Sequence
• Cell Line, Tumor
• Cell Transformation, Neoplastic
• DNA Primers
• Female
• Humans
• Immunohistochemistry
• Male
• Mice
• Mice, Inbred C57BL
• NF-kappa B/metabolism
• Neoplasm Metastasis
• Neoplasms/metabolism
• Neoplasms/pathology
• Precancerous Conditions/metabolism
• Precancerous Conditions/pathology
• Proteins/genetics
• Proteins/physiology
• Proto-Oncogene Proteins c-bcl-2/genetics
• RNA Interference
• RNA, Messenger/genetics
• Reverse Transcriptase Polymerase Chain Reaction
• STAT3 Transcription Factor/genetics
• Stem Cells/metabolism
• Transplantation, Heterologous
• Up-Regulation/physiology

While it is accepted that a majority of invasive breast cancer progresses from a ductal carcinoma in situ (DCIS) precursor stage, very little is known about the factors that promote survival of DCIS neoplastic cells within the hypoxic, nutrient deprived intraductal microenvironment.

We examined the hypothesis that fresh human DCIS lesions contain pre-existing carcinoma precursor cells. We characterized these cells by full genome molecular cytogenetics (Illumina HumanCytoSNP profile), and signal pathway profiling (Reverse Phase Protein Microarray, 59 endpoints), and demonstrated that autophagy is required for survival and anchorage independent growth of the cytogenetically abnormal tumorigenic DCIS cells. Ex vivo organoid culture of fresh human DCIS lesions, without enzymatic treatment or sorting, induced the emergence of neoplastic epithelial cells exhibiting the following characteristics: a) spontaneous generation of hundreds of spheroids and duct-like 3-D structures in culture within 2–4 weeks; b) tumorigenicity in NOD/SCID mice; c) cytogenetically abnormal (copy number loss or gain in chromosomes including 1, 5, 6, 8, 13, 17) compared to the normal karyotype of the non-neoplastic cells in the source patient's breast tissue; d) in vitro migration and invasion of autologous breast stroma; and e) up-regulation of signal pathways linked to, and components of, cellular autophagy. Multiple autophagy markers were present in the patient's original DCIS lesion and the mouse xenograft. We tested whether autophagy was necessary for survival of cytogenetically abnormal DCIS cells. The lysosomotropic inhibitor (chloroquine phosphate) of autophagy completely suppressed the generation of DCIS spheroids/3-D structures, suppressed ex vivo invasion of autologous stroma, induced apoptosis, suppressed autophagy associated proteins including Atg5, AKT/PI3 Kinase and mTOR, eliminated cytogenetically abnormal spheroid forming cells from the organ culture, and abrogated xenograft tumor formation.

Cytogenetically abnormal spheroid forming, tumorigenic, and invasive neoplastic epithelial cells pre-exist in human DCIS and require cellular autophagy for survival.

• Animals
• Autophagy
• Carcinoma, Intraductal, Noninfiltrating/pathology
• Cell Survival
• Chromosome Aberrations
• Genome, Human/genetics
• Humans
• Mice
• Mice, SCID
• Neoplasm Invasiveness
• Neoplastic Stem Cells/pathology
• Neoplastic Stem Cells/transplantation
• Transplantation, Heterologous
• Tumor Cells, Cultured

• Animals
• Antigens, Polyomavirus Transforming/metabolism
• Cell Transformation, Neoplastic/metabolism
• Humans
• Mice
• Mice, Transgenic
• Polyomavirus/immunology
• Polyomavirus/metabolism
• Polyomavirus Infections/pathology
• Polyomavirus Infections/virology
• Signal Transduction
• Tumor Virus Infections/pathology
• Tumor Virus Infections/virology

• P01-CA50661 NCI NIH HHS
• R01-CA29270 NCI NIH HHS
• R01-CA58763 NCI NIH HHS
• R01-CA34722 NCI NIH HHS
• R01 CA034722 NCI NIH HHS
• P01 CA050661 NCI NIH HHS