A permanent window for the murine lung enables high-resolution imaging of cancer metastasis.

Stable, high-resolution intravital imaging of the lung has become possible through the utilization of vacuum-stabilized imaging windows. However, this technique is extremely invasive and limited to only hours in duration. Here we describe a minimally invasive, permanently implantable window for high-resolution intravital imaging of the murine lung that allows the mouse to survive surgery, recover from anesthesia, and breathe independently. Compared to vacuum-stabilized windows, this window produces the same high-quality images without vacuum-induced artifacts; it is also less invasive, which allows imaging of the same lung tissue over a period of weeks. We further adapt the technique of microcartography for reliable relocalization of the same cells longitudinally. Using commonly employed experimental, as well as more clinically relevant, spontaneous metastasis models, we visualize all stages of metastatic seeding, including: tumor cell arrival; extravasation; growth and progression to micrometastases; as well as tumor microenvironment of metastasis function, the hallmark of hematogenous dissemination of tumor cells.

Myosin-IIA heavy chain phosphorylation on S1943 regulates tumor metastasis.

Nonmuscle myosin-IIA (NMHC-IIA) heavy chain phosphorylation has gained recognition as an important feature of myosin-II regulation. In previous work, we showed that phosphorylation on S1943 promotes myosin-IIA filament disassembly in vitro and enhances EGF-stimulated lamellipod extension of breast tumor cells. However, the contribution of NMHC-IIA S1943 phosphorylation to the modulation of invasive cellular behavior and metastasis has not been examined. Stable expression of phosphomimetic (S1943E) or non-phosphorylatable (S1943A) NMHC-IIA in breast cancer cells revealed that S1943 phosphorylation enhances invadopodia function, and is critical for matrix degradation in vitro and experimental metastasis in vivo. These studies demonstrate a novel link between NMHC-IIA S1943 phosphorylation, the regulation of extracellular matrix degradation and tumor cell invasion and metastasis.

Signatures of breast cancer metastasis at a glance.

Gene expression profiling has yielded expression signatures from which prognostic tests can be derived to facilitate clinical decision making in breast cancer patients. Some of these signatures are based on profiling of whole tumor tissue (tissue signatures), which includes all tumor and stromal cells. Prognostic markers have also been derived from the profiling of metastasizing tumor cells, including circulating tumor cells (CTCs) and migratory-disseminating tumor cells within the primary tumor. The metastasis signatures based on CTCs and migratory-disseminating tumor cells have greater potential for unraveling cell biology insights and mechanistic underpinnings of tumor cell dissemination and metastasis. Of clinical interest is the promise that stratification of patients into high or low metastatic risk, as well as assessing the need for cytotoxic therapy, might be improved if prognostics derived from these two types of signatures are used in a combined way. The aim of this Cell Science at a Glance article and accompanying poster is to navigate through both types of signatures and their derived prognostics, as well as to highlight biological insights and clinical applications that could be derived from them, especially when they are used in combination.

Assessment of MRI to estimate metastatic dissemination risk and prometastatic effects of chemotherapy.

Metastatic dissemination in breast cancer is regulated by specialized intravasation sites called "tumor microenvironment of metastasis" (TMEM) doorways, composed of a tumor cell expressing the actin-regulatory protein Mena, a perivascular macrophage, and an endothelial cell, all in stable physical contact. High TMEM doorway number is associated with an increased risk of distant metastasis in human breast cancer and mouse models of breast carcinoma. Here, we developed a novel magnetic resonance imaging (MRI) methodology, called TMEM Activity-MRI, to detect TMEM-associated vascular openings that serve as the portal of entry for cancer cell intravasation and metastatic dissemination. We demonstrate that TMEM Activity-MRI correlates with primary tumor TMEM doorway counts in both breast cancer patients and mouse models, including MMTV-PyMT and patient-derived xenograft models. In addition, TMEM Activity-MRI is reduced in mouse models upon treatment with rebastinib, a specific and potent TMEM doorway inhibitor. TMEM Activity-MRI is an assay that specifically measures TMEM-associated vascular opening (TAVO) events in the tumor microenvironment, and as such, can be utilized in mechanistic studies investigating molecular pathways of cancer cell dissemination and metastasis. Finally, we demonstrate that TMEM Activity-MRI increases upon treatment with paclitaxel in mouse models, consistent with prior observations that chemotherapy enhances TMEM doorway assembly and activity in human breast cancer. Our findings suggest that TMEM Activity-MRI is a promising precision medicine tool for localized breast cancer that could be used as a non-invasive test to determine metastatic risk and serve as an intermediate pharmacodynamic biomarker to monitor therapeutic response to agents that block TMEM doorway-mediated dissemination.

Live tumor imaging shows macrophage induction and TMEM-mediated enrichment of cancer stem cells during metastatic dissemination.

Cancer stem cells (CSCs) play an important role during metastasis, but the dynamic behavior and induction mechanisms of CSCs are not well understood. Here, we employ high-resolution intravital microscopy using a CSC biosensor to directly observe CSCs in live mice with mammary tumors. CSCs display the slow-migratory, invadopod-rich phenotype that is the hallmark of disseminating tumor cells. CSCs are enriched near macrophages, particularly near macrophage-containing intravasation sites called Tumor Microenvironment of Metastasis (TMEM) doorways. Substantial enrichment of CSCs occurs on association with TMEM doorways, contributing to the finding that CSCs represent >60% of circulating tumor cells. Mechanistically, stemness is induced in non-stem cancer cells upon their direct contact with macrophages via Notch-Jagged signaling. In breast cancers from patients, the density of TMEM doorways correlates with the proportion of cancer cells expressing stem cell markers, indicating that in human breast cancer TMEM doorways are not only cancer cell intravasation portals but also CSC programming sites.

Electronic early notification of sepsis in hospitalized ward patients: a study protocol for a stepped-wedge cluster randomized controlled trial.

BACKGROUND: To evaluate the effect of screening for sepsis using an electronic sepsis alert vs. no alert in hospitalized ward patients on 90-day in-hospital mortality. METHODS: The SCREEN trial is designed as a stepped-wedge cluster randomized controlled trial. Hospital wards (total of 45 wards, constituting clusters in this design) are randomized to have active alert vs. masked alert, 5 wards at a time, with each 5 wards constituting a sequence. The study consists of ten 2-month periods with a phased introduction of the intervention. In the first period, all wards have a masked alert for 2 months. Afterwards the intervention (alert system) is implemented in a new sequence every 2-month period until the intervention is implemented in all sequences. The intervention includes the implementation of an electronic alert system developed in the hospital electronic medical records based on the quick sequential organ failure assessment (qSOFA). The alert system sends notifications of "possible sepsis alert" to the bedside nurse, charge nurse, and primary medical team and requires an acknowledgment in the health information system from the bedside nurse and physician. The calculated sample size is 65,250. The primary endpoint is in-hospital mortality by 90 days. DISCUSSION: The trial started on October 1, 2019, and is expected to complete patient follow-up by the end of October 2021. TRIAL REGISTRATION: ClinicalTrials.gov NCT04078594 . Registered on September 6, 2019.

A multi-center prospective cohort study of benign breast disease and risk of subsequent breast cancer.

OBJECTIVE: We used a nested case-control design within a large, multi-center cohort of women who underwent a biopsy for benign breast disease (BBD) to assess the association of broad histologic groupings and specific histologic entities with risk of breast cancer. METHODS: Cases were all women who had a biopsy for BBD and who subsequently developed breast cancer; controls were individually matched to cases and were women with a biopsy for BBD who did not develop breast cancer in the same follow-up interval as that for the cases. After exclusions, 1,239 records (615 cases and 624 controls) were available for analysis. We used conditional logistic regression to estimate odds ratios and 95% confidence intervals (CIs). RESULTS: Relative to non-proliferative BBD/normal pathology, the multivariable-adjusted odds ratio for proliferative lesions without atypia was 1.45 (95% CI 1.10-1.90), and that for atypical hyperplasia was 5.27 (95% CI 2.29-12.15). The presence of multiple foci of columnar cell hyperplasia and of complex fibroadenoma without atypia was associated with a non-significantly increased risk of breast cancer, whereas sclerosing adenosis, radial scar, and papilloma showed no association with risk. CONCLUSION: Our results indicate that, compared to women with normal pathology/non-proliferative disease, women with proliferative disease without atypia have a modestly increased risk of breast cancer, whereas women with atypical hyperplasia have a substantially increased risk.

Tumor microenvironment of metastasis in human breast carcinoma: a potential prognostic marker linked to hematogenous dissemination.

PURPOSE: Multiphoton-based intravital imaging has shown that invasive carcinoma cells in mouse and rat mammary tumors intravasate when associated with perivascular macrophages, identifying a potential tumor microenvironment of metastasis (TMEM). We define TMEM as the tripartite arrangement of an invasive carcinoma cell, a macrophage, and an endothelial cell. The aim of this study was to determine if TMEM density in human breast carcinoma samples predicts the development of systemic, hematogenous metastases. EXPERIMENTAL DESIGN: A case-control study of 30 patients who developed metastatic breast cancer and 30 patients without metastatic disease was done. Cases were matched to controls based on currently used prognostic criteria. Paraffin-embedded primary breast cancer samples were stained using a triple immunohistochemical method allowing simultaneous identification of carcinoma cells, macrophages, and endothelial cells. Two pathologists, blinded to outcome, evaluated the number of TMEM per 20 high-power fields. RESULTS: No association was seen between TMEM density and tumor size or grade, lymph node metastasis, lymphovascular invasion, or hormone receptor status. TMEM density was greater in the group of patients who developed systemic metastases compared with the patients with only localized breast cancer (median, 105 versus 50, respectively; P = 0.00006). For every 10-unit increase in TMEM density, the odds ratio for systemic metastasis was 1.9 (95% confidence interval, 1.1-3.4). CONCLUSIONS: TMEM density predicted the development of systemic, hematogenous metastases. The ability of TMEM to predict distant metastasis was independent of lymph node status and other currently used prognosticators. Quantitation of TMEM may be a useful new prognostic marker for breast cancer patients.

Validation of an Automated Quantitative Digital Pathology Approach for Scoring TMEM, a Prognostic Biomarker for Metastasis.

Metastasis causes ~90% of breast cancer mortality. However, standard prognostic tests based mostly on proliferation genes do not measure metastatic potential. Tumor MicroEnvironment of Metastasis (TMEM), an immunohistochemical biomarker for doorways on blood vessels that support tumor cell dissemination is prognostic for metastatic outcome in breast cancer patients. Studies quantifying TMEM doorways have involved manual scoring by pathologists utilizing static digital microscopy: a labor-intensive process unsuitable for use in clinical practice. We report here a validation study evaluating a new quantitative digital pathology (QDP) tool (TMEM-DP) for identification and quantification of TMEM doorways that closely mimics pathologists' workflow and reduces pathologists' variability to levels suitable for use in a clinical setting. Blinded to outcome, QDP was applied to a nested case-control study consisting of 259 matched case-control pairs. Sixty subjects of these were manually scored by five pathologists, digitally recorded using whole slide imaging (WSI), and then used for algorithm development and optimization. Validation was performed on the remainder of the cohort. TMEM-DP shows excellent reproducibility and concordance and reduces pathologist time from ~60 min to ~5 min per case. Concordance between manual scoring and TMEM-DP was found to be >0.79. These results show that TMEM-DP is capable of accurately identifying and scoring TMEM doorways (also known as MetaSite score) equivalent to pathologists.

Hematogenous Dissemination of Breast Cancer Cells From Lymph Nodes Is Mediated by Tumor MicroEnvironment of Metastasis Doorways.

In primary breast tumors, cancer cells hematogenously disseminate through doorways in the vasculature composed of three-cell complexes (known as Tumor MicroEnvironment of Metastasis) comprising a perivascular macrophage, a tumor cell overexpressing the actin-regulatory protein Mammalian Enabled (Mena), and an endothelial cell, all in direct physical contact. It has been previously shown that once tumor cells establish lymph node metastases in patients, TMEM doorways form in the metastatic tumor cell nests. However, it has not been established if such lymph node-TMEM doorways actively transit tumor cells into the peripheral circulation and on to tertiary sites. To address this question in this short report, we used a mouse model of lymph node metastasis to demonstrate that TMEM doorways: (1) exist in tumor-positive lymph nodes of mice, (2) are restricted to the blood vascular endothelium, (3) serve as a mechanism for further dissemination to peripheral sites such as to the lungs, and (4) their activity can be abrogated by a pharmaceutical intervention. Our data suggest that cancer cell dissemination via TMEM doorways is a common mechanism of breast cancer cell dissemination to distant sites and thus the pharmacological targeting of TMEM may be necessary, even after resection of the primary tumor, to suppress cancer cell dissemination.

Tumor Microenvironment of Metastasis (TMEM) Doorways Are Restricted to the Blood Vessel Endothelium in Both Primary Breast Cancers and Their Lymph Node Metastases.

Cancer cells metastasize from primary tumors to regional lymph nodes and distant sites via the lymphatic and blood vascular systems, respectively. Our prior work has demonstrated that in primary breast tumors, cancer cells utilize a three-cell complex (known as tumor microenvironment of metastasis, or TMEM) composed of a perivascular macrophage, a tumor cell expressing high levels of the actin-regulatory protein mammalian enabled (Mena), and an endothelial cell as functional "doorways" for hematogenous dissemination. Here, we studied a well-annotated case-control cohort of human invasive ductal carcinoma of the breast and metastatic lymph nodes from a separate breast cancer cohort. We demonstrate that in primary breast tumors, blood vessels are always present within tumor cell nests (TCNs) and tumor-associated stroma (TAS), while lymphatic vessels are only occasionally present in TCN and TAS. Furthermore, TMEM doorways not only exist in primary tumors as previously reported but also in lymph node metastases. In addition, we show that TMEM intravasation doorways are restricted to the blood vascular endothelium in both primary tumors and lymph node metastases, suggesting that breast cancer dissemination to distant sites from both primary tumors and metastatic foci in lymph nodes occurs hematogenously at TMEM doorways. TMEMs are very rarely detected at lymphatic vessels and do not confer clinical prognostic significance, indicating they are not participants in TMEM-associated hematogenous dissemination. These findings are consistent with recent observations that hematogenous dissemination from lymph nodes occurs via blood vessels.

Macrophages orchestrate breast cancer early dissemination and metastasis.

Cancer cell dissemination during very early stages of breast cancer proceeds through poorly understood mechanisms. Here we show, in a mouse model of HER2+ breast cancer, that a previously described sub-population of early-evolved cancer cells requires macrophages for early dissemination. Depletion of macrophages specifically during pre-malignant stages reduces early dissemination and also results in reduced metastatic burden at end stages of cancer progression. Mechanistically, we show that, in pre-malignant lesions, CCL2 produced by cancer cells and myeloid cells attracts CD206+/Tie2+ macrophages and induces Wnt-1 upregulation that in turn downregulates E-cadherin junctions in the HER2+ early cancer cells. We also observe macrophage-containing tumor microenvironments of metastasis structures in the pre-malignant lesions that can operate as portals for intravasation. These data support a causal role for macrophages in early dissemination that affects long-term metastasis development much later in cancer progression. A pilot analysis on human specimens revealed intra-epithelial macrophages and loss of E-cadherin junctions in ductal carcinoma in situ, supporting a potential clinical relevance.

Cyclin D1 repression of peroxisome proliferator-activated receptor gamma expression and transactivation.

The cyclin D1 gene is overexpressed in human breast cancers and is required for oncogene-induced tumorigenesis. Peroxisome proliferator-activated receptor gamma (PPAR gamma) is a nuclear receptor selectively activated by ligands of the thiazolidinedione class. PPAR gamma induces hepatic steatosis, and liganded PPAR gamma promotes adipocyte differentiation. Herein, cyclin D1 inhibited ligand-induced PPAR gamma function, transactivation, expression, and promoter activity. PPAR gamma transactivation induced by the ligand BRL49653 was inhibited by cyclin D1 through a pRB- and cdk-independent mechanism, requiring a region predicted to form an helix-loop-helix (HLH) structure. The cyclin D1 HLH region was also required for repression of the PPAR gamma ligand-binding domain linked to a heterologous DNA binding domain. Adipocyte differentiation by PPAR gamma-specific ligands (BRL49653, troglitazone) was enhanced in cyclin D1(-/-) fibroblasts and reversed by retroviral expression of cyclin D1. Homozygous deletion of the cyclin D1 gene, enhanced expression by PPAR gamma ligands of PPAR gamma and PPAR gamma-responsive genes, and cyclin D1(-/-) mice exhibit hepatic steatosis. Finally, reduction of cyclin D1 abundance in vivo using ponasterone-inducible cyclin D1 antisense transgenic mice, increased expression of PPAR gamma in vivo. The inhibition of PPAR gamma function by cyclin D1 is a new mechanism of signal transduction cross talk between PPAR gamma ligands and mitogenic signals that induce cyclin D1.

Neoadjuvant chemotherapy induces breast cancer metastasis through a TMEM-mediated mechanism.

Breast cancer cells disseminate through TIE2/MENACalc/MENAINV-dependent cancer cell intravasation sites, called tumor microenvironment of metastasis (TMEM), which are clinically validated as prognostic markers of metastasis in breast cancer patients. Using fixed tissue and intravital imaging of a PyMT murine model and patient-derived xenografts, we show that chemotherapy increases the density and activity of TMEM sites and Mena expression and promotes distant metastasis. Moreover, in the residual breast cancers of patients treated with neoadjuvant paclitaxel after doxorubicin plus cyclophosphamide, TMEM score and its mechanistically connected MENAINV isoform expression pattern were both increased, suggesting that chemotherapy, despite decreasing tumor size, increases the risk of metastatic dissemination. Chemotherapy-induced TMEM activity and cancer cell dissemination were reversed by either administration of the TIE2 inhibitor rebastinib or knockdown of the MENA gene. Our results indicate that TMEM score increases and MENA isoform expression pattern changes with chemotherapy and can be used in predicting prometastatic changes in response to chemotherapy. Furthermore, inhibitors of TMEM function may improve clinical benefits of chemotherapy in the neoadjuvant setting or in metastatic disease.

A metastasis biomarker (MetaSite Breast™ Score) is associated with distant recurrence in hormone receptor-positive, HER2-negative early-stage breast cancer.

Metastasis is the primary cause of death in early-stage breast cancer. We evaluated the association between a metastasis biomarker, which we call "Tumor Microenviroment of Metastasis" (TMEM), and risk of recurrence. TMEM are microanatomic structures where invasive tumor cells are in direct contact with endothelial cells and macrophages, and which serve as intravasation sites for tumor cells into the circulation. We evaluated primary tumors from 600 patients with Stage I-III breast cancer treated with adjuvant chemotherapy in trial E2197 (NCT00003519), plus endocrine therapy for hormone receptor (HR)+ disease. TMEM were identified and enumerated using an analytically validated, fully automated digital pathology/image analysis method (MetaSite Breast™), hereafter referred to as MetaSite Score (MS). The objectives were to determine the association between MS and distant relapse free interval (DRFI) and relapse free interval (RFI). MS was not associated with tumor size or nodal status, and correlated poorly with Oncotype DX Recurrence Score (r = 0.29) in 297 patients with HR+/HER2- disease. Proportional hazards models revealed a significant positive association between continuous MS and DRFI (p = 0.001) and RFI (p = 0.00006) in HR+/HER2- disease in years 0-5, and by MS tertiles for DRFI (p = 0.04) and RFI (p = 0.01), but not after year 5 or in triple negative or HER2+ disease. Multivariate models in HR+/HER- disease including continuous MS, clinical covariates, and categorical Recurrence Score (<18, 18-30, > 30) showed MS is an independent predictor for 5-year RFI (p = 0.05). MetaSite Score provides prognostic information for early recurrence complementary to clinicopathologic features and Recurrence Score.

The Selective Tie2 Inhibitor Rebastinib Blocks Recruitment and Function of Tie2Hi Macrophages in Breast Cancer and Pancreatic Neuroendocrine Tumors.

Tumor-infiltrating myeloid cells promote tumor progression by mediating angiogenesis, tumor cell intravasation, and metastasis, which can offset the effects of chemotherapy, radiation, and antiangiogenic therapy. Here, we show that the kinase switch control inhibitor rebastinib inhibits Tie2, a tyrosine kinase receptor expressed on endothelial cells and protumoral Tie2-expressing macrophages in mouse models of metastatic cancer. Rebastinib reduces tumor growth and metastasis in an orthotopic mouse model of metastatic mammary carcinoma through reduction of Tie2+ myeloid cell infiltration, antiangiogenic effects, and blockade of tumor cell intravasation mediated by perivascular Tie2Hi/Vegf-AHi macrophages in the tumor microenvironment of metastasis (TMEM). The antitumor effects of rebastinib enhance the efficacy of microtubule inhibiting chemotherapeutic agents, either eribulin or paclitaxel, by reducing tumor volume, metastasis, and improving overall survival. Rebastinib inhibition of angiopoietin/Tie2 signaling impairs multiple pathways in tumor progression mediated by protumoral Tie2+ macrophages, including TMEM-dependent dissemination and angiopoietin/Tie2-dependent angiogenesis. Rebastinib is a promising therapy for achieving Tie2 inhibition in cancer patients. Mol Cancer Ther; 16(11); 2486-501. ©2017 AACR.

Mother nature's disasters and their health effects: a literature review.

Each year global warming and climate changes are affecting Mother Nature, causing extreme weather events to occur (Patz & Khaliq, 2002). The impact of these disasters uproots populations, causes massive infrastructure destruction, and sets the stage for an increase of population mortality and morbidity. Before, during, and after the disaster, nurses provide health care to those in need. Nurses should be aware of the anticipated diseases, traumas, and short- and long-term illnesses that occur following the different types of natural disasters. The purpose of this article is to review existing data and alert nurses to the potential threats they may face following a natural disaster.

GFP expression in the mammary gland for imaging of mammary tumor cells in transgenic mice.

To examine the behavior of tumor cells in tumors developing directly from mammary tissue in transgenic models, we have evaluated transgenic mice expressing green fluorescent protein (GFP). Using the mouse mammary virus promoter (MMTV) to directly drive expression of GFP, we find low levels of fluorescence in the mammary and salivary glands of transgenic animals. Using MMTV-Cre or WAP-Cre in combination with the Cre-activatable CAG-CAT-EGFP construct, we find stronger expression of GFP that is still tissue specific. These animals provide a range of expression of GFP that is suitable for analysis of transgenic mammary tumors and metastases in vivo at the single cell level of resolution.

Characterization of the expression of the pro-metastatic Mena(INV) isoform during breast tumor progression.

Several functionally distinct isoforms of the actin regulatory Mena are produced by alternative splicing during tumor progression. Forced expression of the Mena(INV) isoform drives invasion, intravasation and metastasis. However, the abundance and distribution of endogenously expressed Mena(INV) within primary tumors during progression remain unknown, as most studies to date have only assessed relative mRNA levels from dissociated tumor samples. We have developed a Mena(INV) isoform-specific monoclonal antibody and used it to examine Mena(INV) expression patterns in mouse mammary and human breast tumors. Mena(INV) expression increases during tumor progression and to examine the relationship between Mena(INV) expression and markers for epithelial or mesenchymal status, stemness, stromal cell types and hypoxic regions. Further, while Mena(INV) robustly expressed in vascularized areas of the tumor, it is not confined to cells adjacent to blood vessels. Altogether, these data demonstrate the specificity and utility of the anti-Mena(INV)-isoform specific antibody, and provide the first description of endogenous Mena(INV) protein expression in mouse and human tumors.

Malignant mixed mullerian tumor of the uterus in a patient taking raloxifene.

BACKGROUND: Uterine malignant mixed mesodermal tumor is a rare variant of uterine cancer. Data suggest that tamoxifen is involved in the pathogenesis. We report a case of a women in whom a malignant mixed mesodermal tumor was diagnosed while she was taking raloxifene, which is also a selective estrogen receptor modulator. CASE: A malignant mixed mesodermal tumor was diagnosed in a 64-year-old woman with a bicornuate uterus while she was taking raloxifene for osteoporosis prevention. Diagnosis had been delayed secondary to sampling of the other uterine horn. CONCLUSION: There may be an association between raloxifene and the development of malignant mixed mesodermal tumor. Special attention should be paid when attempting to sample the endometrium in patients with mullerian abnormalities.