Characterization of tumor-associated macrophages in prostate cancer transgenic mouse models.

BACKGROUND: Tumor-associated macrophages (TAMs) are critical components of the tumor microenvironment (TME) in prostate cancer. Commonly used orthotopic models do not accurately reflect the complete TME of a human patient or the natural initiation and progression of a tumor. Therefore, genetically engineered mouse models are essential for studying the TME as well as advancing TAM-targeted therapies. Two common transgenic (TG) models of prostate cancer are Hi-Myc and transgenic adenocarcinoma of the mouse prostate (TRAMP), but the TME and TAM characteristics of these models have not been well characterized. METHODS: To advance the Hi-Myc and TRAMP models as tools for TAM studies, macrophage infiltration and characteristics were assessed using histopathologic, flow cytometric, and expression analyses in these models at various timepoints during tumor development and progression. RESULTS: In both Hi-Myc and TRAMP models, macrophages adopt a more pro-tumor phenotype in higher histological grade tumors and in older prostate tissue. However, the Hi-Myc and TRAMP prostates differ in their macrophage density, with Hi-Myc tumors exhibiting increased macrophage density and TRAMP tumors exhibiting decreased macrophage density compared to age-matched wild type mice. CONCLUSIONS: The macrophage density and the adenocarcinoma cancer subtype of Hi-Myc appear to better mirror patient tumors, suggesting that the Hi-Myc model is the more appropriate in vivo TG model for studying TAMs and TME-targeted therapies.

Understanding the tumor-immune microenvironment in prostate cancer.

PURPOSE OF REVIEW: This review aims to highlight recent advances in prostate cancer tumor-immune microenvironment research and summarize the state-of-the-art knowledge of immune checkpoint inhibitors in prostate cancer. RECENT FINDINGS: Immune checkpoint inhibitors are the cornerstone of modern immunotherapy which have shown encouraging results across a spectrum of cancers. However, only limited survival benefit has been seen in patients with prostate cancer. Prostate cancer progression and its response to immunotherapies are strongly influenced by the tumor-immune microenvironment, whose feature can be summarized as low amounts of tumor-specific antigens, low frequency of tumor-infiltrating lymphocytes and high frequency of tumor-associated macrophages. To improve the therapeutic effect of immunotherapies, in recent years, many strategies have been applied, of which the most promising ones include the combination of multiple immunotherapeutic agents, the combination of an immunotherapeutic agent with other modalities in parallel or in sequential, and the development of biomarkers to find a subgroup of patients who may benefit the most from immunotherapeutic agents. SUMMARY: The impact of immune content and specific immune cell types on prostate cancer biology is highly complex. Recent clinical trials have shed light on the optimal use of immunotherapies for prostate cancer.

Cancer Cells and M2 Macrophages: Cooperative Invasive Ecosystem Engineers.

Many aspects of cancer can be explained utilizing well-defined ecological principles. Applying these principles to cancer, cancer cells are an invasive species to a healthy organ ecosystem. In their capacity as ecosystem engineers, cancer cells release cytokines that recruit monocytes to the tumor and polarize them to M2-like protumor macrophages. Macrophages, recruited by the cancer cells, act as a secondary invasive species. The ecosystem engineering functions of M2-macrophages in turn support and stimulate cancer cell survival and proliferation. The cooperative ecosystem engineering of both the primary invasive species of the cancer cell and the secondary invasive species of the M2-macrophage thus creates a vicious cycle of tumor promotion. Targeting a specific aspect of this tumor-promoting ecosystem engineering, such as blocking efferocytosis by M2-like macrophages, may improve the response to standard-of-care anticancer therapies. This strategy has the potential to redirect cooperative protumor ecosystem engineering toward an antitumor ecosystem engineering strategy.

Targeting Tyro3, Axl and MerTK (TAM receptors): implications for macrophages in the tumor microenvironment.

Tumor-associated macrophages are an abundant cell type in the tumor microenvironment. These macrophages serve as a promising target for treatment of cancer due to their roles in promoting cancer progression and simultaneous immunosuppression. The TAM receptors (Tyro3, Axl and MerTK) are promising therapeutic targets on tumor-associated macrophages. The TAM receptors are a family of receptor tyrosine kinases with shared ligands Gas6 and Protein S that skew macrophage polarization towards a pro-tumor M2-like phenotype. In macrophages, the TAM receptors also promote apoptotic cell clearance, a tumor-promoting process called efferocytosis. The TAM receptors bind the "eat-me" signal phosphatidylserine on apoptotic cell membranes using Gas6 and Protein S as bridging ligands. Post-efferocytosis, macrophages are further polarized to a pro-tumor M2-like phenotype and secrete increased levels of immunosuppressive cytokines. Since M2 polarization and efferocytosis are tumor-promoting processes, the TAM receptors on macrophages serve as exciting targets for cancer therapy. Current TAM receptor-directed therapies in preclinical development and clinical trials may have anti-cancer effects though impacting macrophage phenotype and function in addition to the cancer cells.

The FLRT3-UNC5B checkpoint pathway inhibits T cell-based cancer immunotherapies.

Cancers exploit coinhibitory receptors on T cells to escape tumor immunity, and targeting such mechanisms has shown remarkable clinical benefit, but in a limited subset of patients. We hypothesized that cancer cells mimic noncanonical mechanisms of early development such as axon guidance pathways to evade T cell immunity. Using gain-of-function genetic screens, we profiled axon guidance proteins on human T cells and their cognate ligands and identified fibronectin leucine-rich transmembrane protein 3 (FLRT3) as a ligand that inhibits T cell activity. We demonstrated that FLRT3 inhibits T cells through UNC5B, an axon guidance receptor that is up-regulated on activated human T cells. FLRT3 expressed in human cancers favored tumor growth and inhibited CAR-T and BiTE + T cell killing and infiltration in humanized cancer models. An FLRT3 monoclonal antibody that blocked FLRT3-UNC5B interactions reversed these effects in an immune-dependent manner. This study supports the concept that axon guidance proteins mimic T cell checkpoints and can be targeted for cancer immunotherapy.

Targeting interleukin 4 receptor alpha on tumor-associated macrophages reduces the pro-tumor macrophage phenotype.

Tumor-associated macrophages (TAMs) are an abundant tumor-promoting cell type in the tumor microenvironment (TME). Most TAMs exhibit a pro-tumor M2-like phenotype supportive of tumor growth, immune evasion, and metastasis. IL-4 and IL-13 are major cytokines that polarize macrophages to an M2 subset and share a common receptor, IL-4 receptor alpha (IL-4R alpha). Treatment of human ex vivo polarized M2 macrophages and M2 macrophage precursors with IL-4R alpha antagonist antibody Dupilumab (DupixentⓇ) reduces M2 macrophage features, including a shift in cell surface marker protein expression and gene expression. In animal models of prostate cancer, both pharmacologic inhibition of IL-4R alpha and genetic deletion of IL-4R alpha utilizing an Il4ra -/- mouse model result in decreased CD206 on TAMs. These data support IL-4R alpha as a target to reduce the pro-tumor, M2-like macrophage phenotype as a novel adjunct cancer therapy.

Hemodynamic Safety of Continuous Infusion Labetalol Versus Esmolol Combination Therapies for Type B Aortic Dissections.

BACKGROUND: Medical management for type B aortic dissections (TBADs) require aggressive blood pressure and heart rate control to minimize further dissection extension and to restore perfusion to vital organs. Current guidelines recommend ß-blockers (BB) as first-line therapy, however do not differentiate an ideal agent for use. OBJECTIVE: This study evaluated the hemodynamic safety of continuous infusion labetalol compared to esmolol combination (EC) therapies for TBADs. METHODS: This single-center, retrospective analysis identified patients with a TBAD who received high dose continuous intravenous labetalol (HD-CIVL) or EC therapies. Patients who received HD-CIVL or EC therapies for a minimum of 2 hours, during which a minimum of 4 blood-pressure readings were recorded, were included. The primary end point was the incidence of hemodynamic instability with the use of HD-CIVL versus EC therapies. RESULTS: A total of 20 patients receiving HD-CIVL and 22 patients receiving EC therapy were included in the analysis. Ten (50%) of patients receiving HD-CIVL and 7 (32%) of patients receiving EC therapies met the clinical definition of hemodynamic instability (P = .23). Patients experiencing hemodynamic instability were all due to hypotension, with one also being due to bradycardia. Over half the patients in both groups had discontinued therapy ( P = .06) and were administered bolus fluids (P = .27). Only one patient receiving HD-CIVL required vasopressor administration while none in the EC group (P = .48). CONCLUSION: Our study suggests that HD-CIVL is associated with a nonstatistical significant higher incidence of hemodynamic instability compared to an EC regimen in TBADs. Further studies are warranted in this patient population.

It's Just Math-Unless It's Toxic!

OBJECTIVE: The objective was to determine emergency medicine residents' ability to perform pharmaceutical calculations. METHODS: A six-question needs-assessment survey of emergency medicine residents was conducted at a citywide conference in 2016. Residents performed simple pharmaceutical calculations and were queried regarding resources they typically use to aid in performing calculations. RESULTS: Fifty-three emergency medicine residents out of 110 attendees (48%) completed the survey (postgraduate year [PGY]-1 n = 27, PGY-2 n = 13, PGY-3 n = 8, PGY-4 or above n = 5). Nearly 80% (n = 42) of all residents responded correctly to at least four of six questions (PGY-1 = 70%, PGY-2 = 86%, PGY-3 = 88%, and PGY-4 and above = 100%). Sixty-five percent of PGY-1s, 50% of PGY-2s, and 75% of PGY-3s thought that it was very important to correctly perform basic calculations. Google and Up To Date were frequently used to assist with calculations. More than 70% of all residents utilize clinical pharmacy (PharmD) staff for calculating doses. PharmD services were available at all times in the majority of survey responses. CONCLUSIONS: Emergency medicine residents performed poorly when completing pharmaceutical calculations. This may contribute to increased patient morbidity and mortality. Internet resource use increased as the years of training increased. PharmD services were extensively utilized by all resident regardless of years of training.

A Review on the New and Old Anticoagulants.

Anticoagulants serve as the primary strategy for the prevention and treatment of both arterial and venous thromboembolism. Anticoagulants disrupt coagulation by interfering at various points in the coagulation cascade. This class of medications does not lyse clots that already exist; rather, it prevents thrombus formation and prevents or slows the extension of an existing clot. For decades, the standard therapy for patients requiring oral anticoagulation was warfarin. However, due to some of the shortcomings of warfarin, including the need for continuous routine monitoring, longtime onset and offset of anticoagulation effect, major food and drug interactions, and high incidence of bleeding, newer agents, termed direct oral anticoagulants, or DOACs were developed. This article will provide a review of clinically important information regarding the most commonly used anticoagulants and their reversal agents.

Targeting the EMT transcription factor TWIST1 overcomes resistance to EGFR inhibitors in EGFR-mutant non-small-cell lung cancer.

Patients with EGFR-mutant non-small-cell lung cancer (NSCLC) have significantly benefited from the use of EGFR tyrosine kinase inhibitors (TKIs). However, long-term efficacy of these therapies is limited due to de novo resistance (~30%) as well as acquired resistance. Epithelial-mesenchymal transition transcription factors (EMT-TFs), have been identified as drivers of EMT-mediated resistance to EGFR TKIs, however, strategies to target EMT-TFs are lacking. As the third generation EGFR TKI, osimertinib, has now been adopted in the first-line setting, the frequency of T790M mutations will significantly decrease in the acquired resistance setting. Previously less common mechanisms of acquired resistance to first generation EGFR TKIs including EMT are now being observed at an increased frequency after osimertinib. Importantly, there are no other FDA approved targeted therapies after progression on osimertinib. Here, we investigated a novel strategy to overcome EGFR TKI resistance through targeting the EMT-TF, TWIST1, in EGFR-mutant NSCLC. We demonstrated that genetic silencing of TWIST1 or treatment with the TWIST1 inhibitor, harmine, resulted in growth inhibition and apoptosis in EGFR-mutant NSCLC. TWIST1 overexpression resulted in erlotinib and osimertinib resistance in EGFR-mutant NSCLC cells. Conversely, genetic and pharmacological inhibition of TWIST1 in EGFR TKI-resistant EGFR-mutant cells increased sensitivity to EGFR TKIs. TWIST1-mediated EGFR TKI resistance was due in part to TWIST1 suppression of transcription of the pro-apoptotic BH3-only gene, BCL2L11 (BIM), by directly binding to BCL2L11 intronic regions and promoter. As such, pan-BCL2 inhibitor treatment overcame TWIST1-mediated EGFR TKI resistance and were more effective in the setting of TWIST1 overexpression. Finally, in a mouse model of autochthonous EGFR-mutant lung cancer, Twist1 overexpression resulted in erlotinib resistance and suppression of erlotinib-induced apoptosis. These studies establish TWIST1 as a driver of resistance to EGFR TKIs and provide rationale for use of TWIST1 inhibitors or BCL2 inhibitors as means to overcome EMT-mediated resistance to EGFR TKIs.

Hybrid model intermediate between a laboratory and field study: A humane paradigm shift in feline research.

OBJECTIVES: Non-surgical contraceptives are under development to provide accessible, affordable and humane alternatives for the management of free-roaming cat populations. The objective of this project was to develop a research approach for promising non-surgical contraceptives using outbred cats in a simulated free-roaming setting, meeting high standards for both animal welfare and scientific rigor. METHODS: A facility, specially constructed with indoor and outdoor living areas, was approved and regulated as both an animal shelter and a United States Department of Agriculture research facility. Thirty female and five male cats, healthy but at high risk of euthanasia, were recruited from animal shelters and private homes. Guided by a detailed protocol, cats were housed in this facility for up to 18 months after acclimatization. Cats were administered the study product or a placebo, and then entered into a breeding trial. Cats were adopted at the end of the study. A range of methods was used to provide enrichment and balance a natural environment with the need for detailed daily monitoring. RESULTS: Primary study results related to contraceptive safety and efficacy are published separately. Achieving a research model that is an intermediate step between a laboratory and an uncontained free-roaming cat colony was complex. Significant learnings shared in this current publication span: the selection of cats; acclimatization to a simulated colony environment; cat behavioral training during the study and in preparation for adoption; disease management; contract staff and volunteer support; and cat behavior throughout a breeding study. CONCLUSIONS AND RELEVANCE: This model inspires continued movement away from the paradigm of breeding cats for research and instead sources existing cats at risk for euthanasia. The housing and management of the cats elevates research animals' quality of life and provides positive post-study outcomes. While not appropriate for every feline research scenario, this hybrid model (between a laboratory and field study) proved to be a practical, humane and reliable scenario for research requiring a simulated real-world environment.

Effectiveness of GonaCon as an immunocontraceptive in colony-housed cats.

Objectives Non-surgical contraceptive management of free-roaming cat populations is a global goal for public health and humane reasons. The objectives of this study were to measure the duration of contraception following a single intramuscular injection of a gonadotropin-releasing hormone-based vaccine (GonaCon) and to confirm its safe use in female cats living in colony conditions. Methods GonaCon (0.5 ml/cat) was administered intramuscularly to 20 intact female cats (queens), and saline was administered to 10 queens serving as sham-treated controls. Beginning in late February, 4 months after injection, all cats were housed with fertile male cats in a simulated colony environment. Time to pregnancy, fetal counts and vaccine-elicited injection-site reactions were evaluated. Results All control cats (n = 10/10) and 60% (n = 12/20) of vaccinated cats became pregnant within 4 months of the introduction of males. Two additional vaccinates became pregnant (70%; n = 14/20) within 1 year of treatment. Average fetal counts were significantly lower in vaccinated cats than in control cats. Vaccinates had a significantly longer ( P = 0.0120) median time to conception (212 days) compared with controls (127.5 days). Injection-site reactions ranging from swelling to transient granulomatous masses were observed in 45% (n = 9/20) of vaccinated cats. Conclusions and relevance A single dose of GonaCon provided contraception lasting for a minimum of 1 year in 30% (n = 6/20) of treated cats. The level of contraception induced by this GonaCon dose and vaccine lot was not sufficiently effective to be recommended for use in free-roaming cats.