Extracellular vesicles from young women's breast cancer patients drive increased invasion of non-malignant cells via the Focal Adhesion Kinase pathway: a proteomic approach.

BACKGROUND: Extracellular vesicles (EVs) are small membrane particles that contribute to cancer progression and metastases by transporting biologically significant proteins and nucleic acids. They may also serve as biomarkers of various disease states or important therapeutic targets. Breast cancer EVs have the potential to change the behavior of other cells in their microenvironment. However, the proteomic content of EVs isolated from young women's breast cancer patients and the mechanisms underlying the influence of EVs on tumor cell behavior have not yet been reported. METHODS: In our current translational studies, we compared the proteomic content of EVs isolated from invasive breast cancer cell lines and plasma samples from young women's breast cancer (YWBC) patients and age-matched healthy donors using mass spectrometry. We analyzed the functionality of EVs in two dimensional tumor cell invasion assays and the gene expression changes in tumor cells after incubation with EVs. RESULTS: We found that treatment with EVs from both invasive breast cancer cell lines and plasma of YWBC patients altered the invasive properties of non-invasive breast cancer cells. Proteomics identified differences between EVs from YWBC patients and healthy donors that correlated with their altered function. Further, we identified gene expression changes in non-invasive breast cancer cells after treatment with EVs that implicate the Focal Adhesion Kinase (FAK) signaling pathway as a potential targetable pathway affected by breast cancer-derived EVs. CONCLUSIONS: Our results suggest that the proteome of EVs from breast cancer patients reflects their functionality in tumor motility assays and may help elucidate the role of EVs in breast cancer progression.

Underage purchasing of alcohol from packaged liquor outlets: an Australian study.

Access to the supply of alcohol is an important factor influencing adolescent alcohol consumption. Although alcohol sales outlets are prohibited from selling alcohol to underage youth, there has been limited research investigating compliance. The present study sought to estimate the extent to which adolescents that appeared underage were successfully able to purchase alcohol from packaged liquor outlets in Australia; and to identify store and sales characteristics associated with illegal purchasing. In 2012, purchase surveys were conducted (n= 310) at packaged liquor outlets in 28 urban and rural communities across three states of Australia: Western Australia, Queensland and Victoria. Confederates successfully purchased alcohol at 60% (95% CI: 55-66) of outlets. The density of general alcohol outlets in the surrounding area and the type of liquor outlet were predictors of successful alcohol purchases; however, this was moderated by the state in which the purchase was made. Regional geographical location was also found to predict underage alcohol purchase. The majority of alcohol sales outlets in Australia breach regulations prohibiting sales to underage youth. Consistent enforcement of policies across the states of Australia, and reducing the number of alcohol outlets, will help prevent alcohol outlets illegally selling alcohol to underage adolescents.

Practice patterns in the management of pediatric iliofemoral arterial thrombosis.

BACKGROUND: Acute arterial thrombosis can be life- and limb-threatening. Most pediatric patients with iliofemoral arterial thrombosis are treated successfully with medical therapy; however, expert consensus is limited, and many recommendations are based on the extrapolation of adult data. We aim to understand treatment patterns and long-term outcomes after pediatric acute iliofemoral arterial thrombosis, from which management recommendations can be informed. METHODS: A single-institution retrospective study of pediatric patients diagnosed with iliofemoral arterial thrombosis from 2009 to 2018 was performed. Multiple parameters of management and follow-up were evaluated. Children anticoagulated for ≤28 days versus >28 days were compared. Data analysis used Fisher exact and Mann-Whitney U tests. RESULTS: Two hundred thirty-six children were included. Median age at diagnosis was 65 days (interquartile range 17-163), with 207 diagnosed as infants, 15 diagnosed between 1 to 2 years, and 14 diagnosed between 2 to 16 years. The median treatment duration was 28 days (interquartile range 13-42); patients treated for >28 days had a longer time for thrombus resolution, and more follow-up ultrasounds were performed. Limb length discrepancy did not differ between the groups (1.0% vs 6.3%, P = .06), and no patients were documented to have developed peripheral arterial disease over a median 6.5-year follow-up. Multiple treatment strategies were employed, the most common being heparin bridged to enoxaparin (25.0%) and enoxaparin monotherapy (21.6%). Eight patients (3.4%) underwent surgical intervention. CONCLUSION: Pediatric iliofemoral arterial thrombosis is primarily a disease of infants treated adequately with heparin or enoxaparin, infrequently requires surgical intervention, and is rarely associated with long-term complications. When guided by thrombus resolution on ultrasound, a four-week or shorter course of anticoagulation does not increase the need for surgical intervention or long-term complications.

Triple Targeting of Breast Tumors Driven by Hormonal Receptors and HER2.

Breast cancers that express hormonal receptors (HR) and HER2 display resistance to targeted therapy. Tumor-promotional signaling from the HER2 and estrogen receptor (ER) pathways converges at the cyclin D1 and cyclin-dependent kinases (CDK) 4 and 6 complex, which drives cell-cycle progression and development of therapeutic resistance. Therefore, we hypothesized that co-targeting of ER, HER2, and CDK4/6 may result in improved tumoricidal activity and suppress drug-resistant subclones that arise on therapy. We tested the activity of the triple targeted combination therapy with tucatinib (HER2 small-molecule inhibitor), palbociclib (CKD4/6 inhibitor), and fulvestrant (selective ER degrader) in HR+/HER2+ human breast tumor cell lines and xenograft models. In addition, we evaluated whether triple targeted combination prevents growth of tucatinib or palbociclib-resistant subclones in vitro and in vivo Triple targeted combination significantly reduced HR+/HER2+ tumor cell viability, clonogenic survival, and in vivo growth. Moreover, survival of HR+/HER2+ cells that were resistant to the third drug in the regimen was reduced by the other two drugs in combination. We propose that a targeted triple combination approach will be clinically effective in the treatment of otherwise drug-resistant tumors, inducing robust responses in patients.

An Innovative Model of Urgent Care for the Underserved: A Case Report of an Urban Hospital-Affiliated Federally Qualified Health Center Urgent Care Clinic.

Medically underserved patients experience significant barriers to affordable acute care. This brief report describes an innovative urban hospital-affiliated federally qualified health center urgent care clinic that successfully meets a medically underserved population's need for accessible, appropriate, and affordable acute care.

Defining the Cardiac Fibroblast Secretome in a Fibrotic Microenvironment.

Background Cardiac fibroblasts (CFs) have the ability to sense stiffness changes and respond to biochemical cues to modulate their states as either quiescent or activated myofibroblasts. Given the potential for secretion of bioactive molecules to modulate the cardiac microenvironment, we sought to determine how the CF secretome changes with matrix stiffness and biochemical cues and how this affects cardiac myocytes via paracrine signaling. Methods and Results Myofibroblast activation was modulated in vitro by combining stiffness cues with TGFß1 (transforming growth factor ß 1) treatment using engineered poly (ethylene glycol) hydrogels, and in vivo with isoproterenol treatment. Stiffness, TGFß1, and isoproterenol treatment increased AKT (protein kinase B) phosphorylation, indicating that this pathway may be central to myofibroblast activation regardless of the treatment. Although activation of AKT was shared, different activating cues had distinct effects on downstream cytokine secretion, indicating that not all activated myofibroblasts share the same secretome. To test the effect of cytokines present in the CF secretome on paracrine signaling, neonatal rat ventricular cardiomyocytes were treated with CF conditioned media. Conditioned media from myofibroblasts cultured on stiff substrates and activated by TGFß1 caused hypertrophy, and one of the cytokines in that media was insulin growth factor 1, which is a known mediator of cardiac myocyte hypertrophy. Conclusions Culturing CFs on stiff substrates, treating with TGFß1, and in vivo treatment with isoproterenol all caused myofibroblast activation. Each cue had distinct effects on the secretome or genes encoding the secretome, but only the secretome of activated myofibroblasts on stiff substrates treated with TGFß1 caused myocyte hypertrophy, most likely through insulin growth factor 1.

Epithelial-mesenchymal crosstalk influences cellular behavior in a 3D alveolus-fibroblast model system.

Interactions between lung epithelium and interstitial fibroblasts are increasingly recognized as playing a major role in the progression of several lung pathologies, including cancer. Three-dimensional in vitro co-culture systems offer tissue-relevant platforms to study the signaling interplay between diseased and healthy cell types. Such systems provide a controlled environment in which to probe the mechanisms involved in epithelial-mesenchymal crosstalk. To recapitulate the native alveolar tissue architecture, we employed a cyst templating technique to culture alveolar epithelial cells on photodegradable microspheres and subsequently encapsulated the cell-laden spheres within poly (ethylene glycol) (PEG) hydrogels containing dispersed pulmonary fibroblasts. A fibroblast cell line (CCL-210) was co-cultured with either healthy mouse alveolar epithelial primary cells or a cancerous alveolar epithelial cell line (A549) to probe the influence of tumor-stromal interactions on proliferation, migration, and matrix remodeling. In 3D co-culture, cancerous epithelial cells and fibroblasts had higher proliferation rates. When examining fibroblast motility, the fibroblasts migrated faster when co-cultured with cancerous A549 cells. Finally, a fluorescent peptide reporter for matrix metalloproteinase (MMP) activity revealed increased MMP activity when A549s and fibroblasts were co-cultured. When MMP activity was inhibited or when cells were cultured in gels with a non-degradable crosslinker, fibroblast migration was dramatically suppressed, and the increase in cancer cell proliferation in co-culture was abrogated. Together, this evidence supports the idea that there is an exchange between the alveolar epithelium and surrounding fibroblasts during cancer progression that depends on MMP activity and points to potential signaling routes that merit further investigation to determine targets for cancer treatment.