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BACKGROUND: Metastatic triple-negative breast cancer (mTNBC) is treated mainly with chemotherapy. However, resistance frequently occurs as tumours enter dormancy. Statins have been suggested as effective against cancer but as they prolong and promote dormancy, it is an open question of whether the concomitant use would interfere with chemotherapy in primary and mTNBC. We examined this question in animal models and clinical correlations. METHODS: We used a xenograft model of spontaneous metastasis to the liver from an ectopic tumour employing a mTNBC cell line. Atorvastatin was provided to sensitise metastatic cells, followed by chemotherapy. The effects of statin usage on outcomes in women with metastatic breast cancer was assessed respectively by querying a database of those diagnosed from 1999 to 2019. RESULTS: Atorvastatin had limited influence on tumour growth or chemotherapy effects in ectopic primary tumours. Interestingly, atorvastatin was additive with doxorubicin (but not paclitaxel) when targeting liver metastases. E-cadherin-expressing, dormant, breast cancer cells were resistant to the use of either statins or chemotherapy as compared to wild-type cells; however, the combination of both did lead to increased cell death. Although prospective randomised studies are needed for validation, our retrospective clinical analysis suggested that patients on statin treatment could experience prolonged dormancy and overall survival; still once the tumour recurred progression was not affected by statin use. CONCLUSION: Atorvastatin could be used during adjuvant chemotherapy and also in conjunction with metastatic chemotherapy to reduce mTNBC cancer progression. These preclinical data establish a rationale for the development of randomised studies.

Wound healing is a complex sequence of tissue protection, replacement, and reorganization leading to regenerated tissue. Disruption of any of these steps results in the process being incomplete as an ulcer or over-exuberant as a hypertrophic scar. Over the past decade, it has become evident that the extracellular matrix and associated components orchestrate this process. However, the cellular events that are induced by the extracellular matrix to accomplish wound healing remain to be defined. Herein we propose that matrix-regulated cellular macro-autophagy is key to both the tissue replacement and resolution stages of healing by directing cellular function or apoptosis. Further, disruptions in matrix turnover alter autophagic function leading to chronic wounds or scarring. While the literature that directly investigates autophagy during wound healing is sparse, the emerging picture supports our proposing a model of the centrality of the matrix-autophagy modulation as central to physiologic and pathologic healing.

The liver is the most commonly involved organ in metastases from a wide variety of solid tumors. The use of biologically and cellularly complex liver tissue systems have shown that tumor cell behavior and therapeutic responses are modulated within the liver microenvironment and in ways distinct from the behaviors in the primary locations. These microphysiological systems have provided unexpected and powerful insights into the tumor cell biology of metastasis. However, neither the tumor nor the liver exist in an isolated tissue situation, having to function within a complete body and respond to systemic events as well as those in other organs. To examine the influence of one organ on the function of other tissues, microphysiological systems are being linked. Herein, we discuss extending this concept to tumor metastases by integrating complex models of the primary tumor with the liver metastatic environment. In addition, inflammatory organs and the immune system can be incorporated into these multi-organ systems to probe the effects on tumor behavior and cancer treatments.

Background: Micrometastases from breast cancer (BC) might undergo quiescence following adjuvant therapies. Oftentimes they reactivate, prompting metastatic relapse and aggressive, uncurable disease. It is necessary to find alternative, safe therapies that could be used as adjuvants for delaying the dormancy process and reduce metastatic breast cancer (MBC) progression. Prior epidemiological studies found prolonged overall survival from chronic statin use. However, the effectiveness of statin use through different stages of progression, including advanced MBC, has not yet been defined. Methods: A real-world, retrospective analysis of 1,016 deceased patients with MBC treated from 1999-2019 at the University of Pittsburgh Medical Center (UPMC). Lipophilic statin (atorvastatin and simvastatin) use and dosing were retrieved from medical records through an IRB-approved R3 request and merged with already available clinical variables. A total of 122 patients were included in the analysis as the statin use group. This included patients who were prescribed statins through the course of their breast cancer. For measuring time to relapse, only patients with statin prescription before being diagnosed with MBC were selected. Log-ranks were used to compute survival. Uni- and multi-variate Cox hazard models were also applied. Statistical analysis was computed using the package ggpubr on R v3.6.3.Results: Women who used statins were older at the time of detection of primary (60 vs 52 years of age, respectively) and MBC (66 vs 56 years of age, respectively). An almost 2-fold reduction was found in the onset of liver metastasis as first relapse in the statin group, compared to general population (5.7% vs 10.4%, respectively). Increased overall survival (OS) was found among statin users who had ER (+) (p Citation Format: Juan Luis Gomez Marti, Alan Wells. Lipophilic statins for prolonging dormancy and overall survival: A real-world retrospective investigation [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS11-22.

Background Even though we have established a few risk factors for metastatic breast cancer (MBC) through epidemiologic studies, these risk factors have not proven to be effective in predicting an individual’s risk of developing metastasis. Therefore, identifying critical risk factors for MBC continues to be a major research imperative, and one which can lead to advances in breast cancer clinical care. The objective of this research is to leverage Bayesian Networks (BN) and information theory to identify key risk factors for breast cancer metastasis from data. Methods We develop the Markov Blanket and Interactive risk factor Learner (MBIL) algorithm, which learns single and interactive risk factors having a direct influence on a patient’s outcome. We evaluate the effectiveness of MBIL using simulated datasets, and compare MBIL with the BN learning algorithms Fast Greedy Search (FGS), PC algorithm (PC), and CPC algorithm (CPC). We apply MBIL to learn risk factors for 5 year breast cancer metastasis using a clinical dataset we curated. We evaluate the learned risk factors by consulting with breast cancer experts and literature. We further evaluate the effectiveness of MBIL at learning risk factors for breast cancer metastasis by comparing it to the BN learning algorithms Necessary Path Condition (NPC) and Greedy Equivalent Search (GES). Results The averages of the Jaccard index for the simulated datasets containing 2000 records were 0.705, 0.272, 0.228, and 0.147 for MBIL, FGS, PC, and CPC respectively. MBIL, NPC, and GES all learned that grade and lymph_nodes_positive are direct risk factors for 5 year metastasis. Only MBIL and NPC found that surgical_margins is a direct risk factor. Only NPC found that invasive is a direct risk factor. MBIL learned that HER2 and ER interact to directly affect 5 year metastasis. Neither GES nor NPC learned that HER2 and ER are direct risk factors. Discussion The results involving simulated datasets indicated that MBIL can learn direct risk factors substantially better than standard Bayesian network learning algorithms. An application of MBIL to a real breast cancer dataset identified both single and interactive risk factors that directly influence breast cancer metastasis, which can be investigated further.

BACKGROUND: Antidepressant use later in pregnancy has been associated with preeclampsia and postpartum hemorrhage (PPH) in some studies. OBJECTIVE: To evaluate the association between patterns of prenatal antidepressant dose across gestation and risk of precclampsia and PPH. METHODS: We utilized OptumLabs® Data Warehouse (2012–2016) administrative health care claims, identifying 226,932 singleton live-born deliveries for this retrospective cohort study. Antidepressant dispensing doses were converted to fluoxetine equivalents. Using k-means longitudinal, we identified women with similar patterns of antidepressant exposure, i.e., trajectory groups, during the first 20 and 35 gestational weeks. We estimated risk ratios (RR) and 95% confidence intervals (CI) for the association between trajectory groups and preeclampisa (20-week groups) and PPH (35-week groups), adjusting for demographics, comorbidities, and other psychotropic medications. Linear trend tests assessing increasing risk of the outcomes across groups were performed. RESULTS: Among 15,041 (6.6%) pregnancies exposed to an antidepressant, the following trajectory groups were identified: A-low exposure, starting pregnancy at ~10mg/day, with 1(st) trimester reduction/discontinuation, B-low sustained exposure of ~20 mg/day, C-moderate exposure (~40mg/day) with 1(st) trimester reduction/discontinuation, D-moderate sustained exposure of ~40 mg/day, and E-high sustained exposure of ~75mg/day. In the low exposure with reduction/discontinuation trajectory, risks were 8.2% for preeclampsia and 2.7% for PPH. Compared with this group, low, moderate, and high sustained trajectories were associated with preeclampsia [adjusted (a)RR: 1.17 (95% CI: 1.01, 1.34), aRR: 1.31 (95% CI: 1.12, 1.54), aRR: 1.41 (95% CI: 1.05, 1.90), respectively] and PPH [aRR: 1.32 (1.05, 1.66), aRR: 1.35 (95% CI: 1.03,1.78), aRR: 2.51 (95% CI: 1.69, 3.71), respectively]; p

Cancer metastasis is the leading cause of mortality in patients with solid tumors. The majority of these deaths are associated with metastatic disease that occurs after a period of clinical remission, anywhere from months to decades following removal of the primary mass. This dormancy is prominent in cancers of the breast and prostate among others, leaving the survivors uncertain about their longer-term prognosis. The most daunting aspect of this dormancy and re-emergence is that the micrometastases in particular, and even large lethal outgrowths are often show resistance to agents to which they have not been exposed. This suggests that in addition to specific mutations that target single agents, there also exist adaptive mechanisms that provide this pan-resistance. Potential molecular underpinnings of which are the topic of this review.

Background: Risk of metastatic recurrence of breast cancer after initial diagnosis and treatment depends on the presence of a number of risk factors. Although most univariate risk factors have been identified using classical methods, machine-learning methods are also being conducted to tease out non-obvious contributors to a patient’s individual risk of developing late distant metastasis. Bayesian-network algorithms may predict not only risk factors but also interactions among these risks, which consequently lead to metastatic breast cancer. We proposed to apply a previously developed machine-learning method to predict risk factors of 5-, 10- and 15-year metastasis. Methods: We applied a previously validated algorithm named the Markov Blanket and Interactive risk factor Learner (MBIL) on the electronic health record (EHR)-based Lynn Sage database (LSDB) from the Lynn Sage Comprehensive Breast Cancer at Northwestern Memorial Hospital. This algorithm provided an output of both single and interactive risk factors of 5-, 10-, and 15-year metastasis from LSDB. We individually examined and interpreted the clinical relevance of these interactions based on years to metastasis and the reliance on interactivity between risk factors. Results: We found that with lower alpha values (low interactivity score), the prevalence of variables with an independent influence on long term metastasis was higher (i.e., HER2, TNEG). As the value of alpha increased to 480, stronger interactions were needed to define clusters of factors that increased the risk of metastasis (i.e., ER, smoking, race, alcohol usage). Conclusion: MBIL identified single and interacting risk factors of metastatic breast cancer, many of which were supported by clinical evidence. These results strongly recommend the development of further large data studies with different databases to validate the degree to which some of these variables impact metastatic breast cancer in the long term.

Objective: Early prostate cancer micrometastatic foci undergo a mesenchymal to epithelial reverting transition, not only aiding seeding and colonization, but also rendering the tumor cells generally chemoresistant. We previously found that upregulated E-cadherin in the epithelial micrometastases activated canonical survival pathways, including PI3K-Akt, that protected the tumor cells from death; however, the extent of protection from blocking the pathway in its entirety was modest, because different isoforms may have alternately affected cell functioning. Here, we characterized Akt isoform expressions in primary and metastatic prostate cancers, as well as their individual contributions to chemoresistance. Methods: Akt isoforms and E-cadherin were manipulated with drugs, knocked down, and over expressed. Tumor cell killing was determined in vitro and in vivo. Overall survival was calculated from patient records and specimens. Results: Pan-Akt inhibition sensitized tumor cells to chemotherapy, and specific blockade of Akt1 or/and Akt2 caused cells to be more chemoresponsive. Overexpression of Akt3 induced apoptosis. A low dose of Akt1 or Akt2 inhibitor enabled standard chemotherapies to significantly eradicate metastatic prostate tumors in a mouse model, acting as chemosensitizers. In human specimens, we found Akt1 and Akt2 positively correlated, whereas Akt3 inversely correlated, with the overall survival of prostate cancer patients. Akt1high/Akt2high/Akt3low tumors had the worst outcomes. Conclusions: E-cadherin-induced activation of Akt1/2 isoforms was the essential mechanism of chemoresistance, whereas Akt3 made cells more fragile. These findings emphasized the need to target Akt1/2, rather than pan-Akt, as a rational therapeutic approach.

Interferon-γ (IFNγ) is a cytokine with limited evidence of benefit in cancer clinical trials to date. However, it could potentially play a role in potentiating anti-tumor immunity in the immunologically "cold" metastatic castration-resistant prostate cancer (mCRPC) by inducing antigen presentation pathways and concurrently providing targets for immune checkpoint blockade therapy. Moreover, it could additionally increase sensitivity to chemotherapy based on its pleiotropic effects on cell phenotype. Here, we show that IFNγ treatment induced expression of major histocompatibility class-I (MHC-I) genes and PD-L1 in prostate cancer cells in vitro. Furthermore, IFNγ treatment led to a decrease in E-cadherin expression with a consequent increase in sensitivity to chemotherapy in vitro. In an in vivo murine tumor model of spontaneous metastatic prostate cancer, IFNγ systemic pretreatment upregulated the expression of HLA-A and decreased E-cadherin expression in the primary tumor, and more importantly in the metastatic site led to increased apoptosis and limited micrometastases in combination with paclitaxel treatment compared to diffuse metastatic disease in control and monotherapy treatment groups. These findings suggest that IFNγ may be useful in combinatorial regimens to induce sensitivity to immunotherapy and chemotherapy in hepatic metastases of mCRPC.

Background Evidence suggests that cytokine imbalances may be at the root of deficits that occur in numerous neurodevelopmental disorders, including schizophrenia and autism spectrum disorder. Notably, while clinical studies have demonstrated maternal cytokine imbalances with alcohol consumption during pregnancy—and data from animal models have identified immune disturbances in alcohol-exposed offspring—to date, immune alterations in alcohol-exposed children have not been explored. Thus, here we hypothesized that perturbations in the immune environment as a result of prenatal alcohol exposure will program the developing immune system, and result in immune dysfunction into childhood. Due to the important role of cytokines in brain development/function, we further hypothesized that child immune profiles might be associated with their neurodevelopmental status. Methods As part of a longitudinal study in Ukraine, children of mothers reporting low/no alcohol consumption or moderate-to-heavy alcohol consumption during pregnancy were enrolled in the study and received neurodevelopmental assessments. Group stratification was based on maternal alcohol consumption and child neurodevelopmental status resulting in the following groups: A/TD, alcohol-consuming mother, typically developing child; A/ND, alcohol-consuming mother, neurodevelopmental delay in the child; C/TD, control mother (low/no alcohol consumption), typically development child; and C/ND, control mother, neurodevelopmental delay in the child. Forty cytokines/chemokines were measured in plasma and data were analyzed using regression and constrained principle component analysis. Results Analyses revealed differential cytokine network activity associated with both prenatal alcohol exposure and neurodevelopmental status. Specifically, alcohol-exposed children showed activation of a cytokine network including eotaxin-3, eotaxin, and bFGF, irrespective of neurodevelopmental status. However, another cytokine network was differentially activated based on neurodevelopmental outcome: A/TD showed activation of MIP-1β, MDC, and MCP-4, and inhibition of CRP and PlGF, with opposing pattern of activation/inhibition detected in the A/ND group. By contrast, in the absence of alcohol-exposure, activation of a network including IL-2, TNF-β, IL-10, and IL-15 was associated with neurodevelopmental delay. Conclusions Taken together, this comprehensive assessment of immune markers allowed for the identification of unique immune milieus that are associated with alcohol exposure as well as both alcohol-related and alcohol-independent neurodevelopmental delay. These findings are a critical step towards establishing unique immune biomarkers for alcohol-related and alcohol-independent neurodevelopmental delay.

BackgroundCarcinoma cells shift between epithelial and mesenchymal phenotypes during cancer progression, as defined by surface presentation of the cell-cell cohesion molecule E-cadherin, affecting dissemination, progression and therapy responsiveness. Concomitant with the loss of E-cadherin during the mesenchymal transition, the predominant receptor isoform for ELR-negative CXC ligands shifts from CXCR3-B to CXCR3-A which turns this classical G-protein coupled receptor from an inhibitor to an activator of cell migration, thus promoting tumor cell invasiveness. We proposed that CXCR3 was not just a coordinately changed receptor but actually a regulator of the cell phenotype.MethodsImmunoblotting, immunofluorescence, quantitative real-time PCR and flow cytometry assays investigated the expression of E-cadherin and CXCR3 isoforms. Intrasplenic inoculation of human prostate cancer (PCa) cells with spontaneous metastasis to the liver analyzed E-cadherin and CXCR3-B expression during cancer progression in vivo.ResultsWe found reciprocal regulation of E-cadherin and CXCR3 isoforms. E-cadherin surface expression promoted CXCR3-B presentation on the cell membrane, and to a lesser extent increased its mRNA and total protein levels. In turn, forced expression of CXCR3-A reduced E-cadherin expression level, whereas CXCR3-B increased E-cadherin in PCa. Meanwhile, a positive correlation of E-cadherin and CXCR3-B expression was found both in experimental PCa liver micro-metastases and patients’ tissue.ConclusionsCXCR3-B and E-cadherin positively correlated in vitro and in vivo in PCa cells and liver metastases, whereas CXCR3-A negatively regulated E-cadherin expression. These results suggest that CXCR3 isoforms may play important roles in cancer progression and dissemination via diametrically regulating tumor’s phenotype.

Studies describing SARS-CoV-2 immune responses following mRNA vaccination in hematology malignancy (HM) patients are virtually non-existent. We measured SARS-CoV-2 IgG production in 67 HM patients who received 2 mRNA vaccine doses. We found that 46% of HM patients did not produce antibodies and were therefore vaccine non-responders. Patients with B-cell CLL were at a particularly high risk, as only 23% had detectable antibodies despite the fact that nearly 70% of these patients were not undergoing cancer therapy. HM patients should be counseled about the ongoing risk of COVID-19 despite vaccination. Routine measurement of post-vaccine antibodies in HM patients should be considered. Novel strategies are needed to prevent COVID-19 in these individuals.

Metastatic breast cancer remains a largely incurable and fatal disease with liver involvement bearing the worst prognosis. The danger is compounded by a subset of disseminated tumor cells that may lie dormant for years to decades before re-emerging as clinically detectable metastases. Pathophysiological signals can drive these tumor cells to emerge. Prior studies indicated CXCR3 ligands as being the predominant signals synergistically and significantly unregulated during inflammation in the gut-liver axis. Of the CXCR3 ligands, IP-10 (CXCL10) was the most abundant, correlated significantly with shortened survival of human breast cancer patients with metastatic disease and was highest in those with triple negative (TNBC) disease. Using a complex ex vivo all-human liver microphysiological (MPS) model of dormant-emergent metastatic progression, CXCR3 ligands were found to be elevated in actively growing populations of metastatic TNBC breast cancer cells whereas they remained similar to the tumor-free hepatic niche in those with dormant breast cancer cells. Subsequent stimulation of dormant breast cancer cells in the ex vivo metastatic liver MPS model with IP-10 triggered their emergence in a dose-dependent manner. Emergence was indicated to occur indirectly possibly via activation of the resident liver cells in the surrounding metastatic microenvironment, as stimulation of breast cancer cells with exogenous IP-10 did not significantly change their migratory, invasive or proliferative behavior. The findings reveal that IP-10 is capable of triggering the emergence of dormant breast cancer cells within the liver metastatic niche and identifies the IP-10/CXCR3 as a candidate targetable pathway for rational approaches aimed at maintaining dormancy.

Local anesthetics have been used since ancient times in medical surgeries and aesthetic procedures. Lidocaine is the most commonly used local anesthetic agent. The infiltration techniques include using small caliber needles and low-volume syringes. The conventional needles have three parts: the hub

Metastatic spread represents the leading cause of disease-related mortality among cancer patients. Many cancer patients suffer from metastatic relapse years or even decades after radical surgery for the primary tumor. This clinical phenomenon is explained by the early dissemination of cancer cells followed by a long period of dormancy. Although dormancy could be viewed as a window of opportunity for therapeutic interventions, dormant disseminated cancer cells and micrometastases, as well as emergent outgrowing macrometastases, exhibit a generalized, innate resistance to chemotherapy and even immunotherapy. This therapeutic pan-resistance, on top of other adaptive responses to targeted agents such as acquired mutations and lineage plasticity, underpins the current difficulties in eradicating cancer. In the present review, we attempt to provide a framework to understand the underlying biology of this major issue.

Background The Fetal Alcohol Spectrum Disorders (FASD) are among the most prevalent causes of neurodevelopmental disorders. The diagnosis is based on assessment of prenatal alcohol exposure, specific physical features identified with a dysmorphology examination, and neurobehavioral assessment. Prompt diagnosis of affected children is necessary to provide early intervention services in a timely manner; however, the availability of diagnostic expertise is limited. We propose telemedicine as a valid and reliable mode by which the physical phenotype of FASD can be accurately assessed METHODS: We compared face to face (F2F) physical examinations of the 3 key facial features and the resulting physical phenotype of the Fetal alcohol syndrome (FAS) and partial FAS (pFAS), as well as 12 additional physical features seen more frequently in children with FAS than in the general population in 61 individuals with two different telemedicine (TM) methods. These included Zoom secure connection system (ZOOM), using a smart phone and a tablet Transportable Examination Station (TES) system using a precision camera and a laptop. We measured the percentages of agreement and the Cohen's K coefficient for each comparison. Results Agreements for most physical features and for the physical phenotype of FAS and pFAS were in the "almost perfect" range with some exceptions in the "substantial" range. Imprecision in measurement and subjectivity underlie lower agreement for some features, both F2F and using TM. We identified the optimal conditions for the F2F examinations in order to assure reliability using TM. Conclusions Telemedicine is a valid and reliable method for the examination of the physical features of FAS, that may contribute to greater access to an early diagnosis of FASD in children prenatally exposed to alcohol and/or with characteristic neurobehavioral deficits.

Most persons with fetal alcohol spectrum disorders (FASDs) remain undiagnosed or are diagnosed in later life. To address the need for earlier diagnosis, we previously assessed miRNAs in the blood plasma of pregnant women who were classified as unexposed to alcohol (UE), heavily exposed with affected infants (HEa), or heavily exposed with apparently unaffected infants (HEua). We reported that maternal miRNAs predicted FASD-related growth and psychomotor deficits in infants. Here, we assessed whether fetal sex influenced alterations in maternal circulating miRNAs following prenatal alcohol exposure (PAE). To overcome the loss of statistical power due to disaggregating maternal samples by fetal sex, we adapted a strategy of iterative bootstrap resampling with replacement to assess the stability of statistical parameter estimates. Bootstrap estimates of parametric and effect size tests identified male and female fetal sex-associated maternal miRNA responses to PAE that were not observed in the aggregated sample. Additionally, we observed, in HEa mothers of female, but not male fetuses, a network of co-secreted miRNAs whose expression was linked to miRNAs encoded on the X-chromosome. Interestingly, the number of significant miRNA correlations for the HEua group mothers with female fetuses was intermediate between HEa and UE mothers at mid-pregnancy, but more similar to UE mothers by the end of pregnancy. Collectively, these data show that fetal sex predicts maternal circulating miRNA adaptations, a critical consideration when adopting maternal miRNAs as diagnostic biomarkers. Moreover, a maternal co-secretion network, predominantly in pregnancies with female fetuses, emerged as an index of risk for adverse birth outcomes due to PAE.

Background Glaucoma filtration surgery (GFS) is limited by subconjunctival, episcleral and scleral fibrosis sealing the trabeculectomy and scarring the filtering bleb. Mitomycin-C (MMC) is commonly applied intraoperatively to the subconjunctival and/or intrascleral space to reduce scarring and promotes GFS success but is associated with postoperative scleral melting and bleb leaks. IP-10 peptide (IP-10p), an ELR-negative CXC chemokine mimetic and inhibitor of fibroblast function, may be an alternative or adjunct to current postoperative GFS treatments. This study sought to determine if IP-10p produces histological changes in tissue remodelling, vascularity and fibrosis that enhance bleb survival after GFS. Methods Rabbits underwent tube-assisted filtration surgery on the right eye with either: (a) IP-10p injected into bleb at time of surgery and postoperative days 2, 4 and 7, (b) intraoperative MMC or (c) intraoperative MMC plus IP-10p injected into bleb at time of surgery and postoperative days 2, 4 and 7. Left contralateral eyes were treated with balanced salt solution (BSS). Results IP-10p-treated blebs demonstrated reduced collagen deposition, cellularity and overall reduction of scar formation compared to BSS-control. Bleb vascularity was reduced compared to BSS-control and MMC treatment groups. Additionally, IP-10p/MMC treated eyes demonstrated an increased number of conjunctival goblet cells in bleb histology compared to the dramatic loss seen with MMC treatment alone. Conclusions This study demonstrates that IP-10p significantly reduces histological scarring compared to BSS or MMC alone, does not damage the conjunctiva to the extent of current standards, and may be an alternative or adjunct to MMC for those undergoing GFS.

OBJECTIVES: To estimate the risk of neonatal outcomes from patterns of prenatal antidepressant use. METHODS: From the OptumLabs Data Warehouse, 226 932 singleton deliveries were identified. Antidepressant claims with coverage between the last menstrual period and 35 weeks’ gestation were converted to fluoxetine equivalents, and a longitudinal cluster analysis was performed. Outcomes included major cardiac malformations (11.7 of 1000 births), preterm birth (75.7 of 1000 births), and newborn respiratory distress (54.2 of 1000 births). The lowest trajectory was the primary reference group, and depression and anxiety with no antidepressant claims served as secondary reference groups. RESULTS: From 15 041 (6.6%) pregnancies exposed to an antidepressant, use patterns were best described as (1) low use (∼10 mg/day) with first-trimester reduction, (2) low sustained use (∼20 mg/day), (3) moderate use (∼40 mg/day) with first-trimester reduction, (4) moderate sustained use (∼40 mg/day), and (5) high sustained use (∼75 mg/day). Moderate sustained use increased the risk of major cardiac malformations, although results included the null when compared with depression or anxiety reference groups. Moderate sustained (adjusted risk ratio [RR] 1.31; 95% confidence interval [CI] 1.16–1.49) and high sustained (adjusted RR 1.78; 95% CI 1.48–2.14) trajectories were associated with an increased risk of preterm birth. All 4 trajectories increased the risk of neonatal respiratory distress in a dose-response fashion (adjusted RRs 1.36 [95% CI 1.20–1.50] to 2.23 [95% CI 1.83–2.77]). CONCLUSIONS: Although findings support continuation of the lowest effective dose to treat depression or anxiety, which benefits the mother, they also highlight an increased risk for newborn respiratory distress in all groups and preterm birth at moderate to high sustained doses.

Cancer mortality ensues from metastatic growths. Cancers use two strategies to allow for this unrelenting expansion. The first way is that early metastases are often cryptic or dormant, being invisible to both innate suppressive actions and undetected clinically. Second, both the micrometastases and later clinically lethal growths are resistant to therapies, whether standard chemotherapies, targeted biologics, or even immunotherapies. These two modes of resistance necessitate new approaches to treatments if we are to eliminate mortality from solid tumors. However, to develop such therapeutic strategies, we first need to better understand the cellular behaviors and molecular events that enable the resistances. Herein, we present a comprehensive model of changing methods of avoidance and resistance that occur during tumor progression, and doubly confound treatment by mixing survival strategies throughout the continuum creating moving targets. Melanoma is presented as the model cancer, as it is being targeted by all three types of agents for disseminated disease, with breast and prostate cancer as two other key carcinomas. Impact statement Cancers kill mainly because metastatic disease is resistant to systemic therapies. It was hoped that newer targeted and immunomodulatory interventions could overcome these issues. However, recent findings point to a generalized resistance to elimination imparted by both cancer-intrinsic and -extrinsic changes to provide survival advantages to the disseminated tumor cells. Here, we present a novel conceptual framework for the microenvironmental inputs and changes that contribute to this generalized therapeutic resistance. In addition we address the issues of experimental systems in terms of studying this phenomenon with their advantages and limitations. This is meant to spur studies into this critical aspect of tumor progression that directly leads to cancer mortality.

HMG-CoA reductase (HMGCR) inhibitors, statins, are potent cholesterol reducing drugs that exhibit anti-tumor effects in vitro and in animal models, including attenuation of metastasis formation, and their use correlates with reduced cancer-specific mortality in retrospective human cohort studies. However, E-cadherin expressing epithelial- and mixed epithelial-mesenchymal cancer cell lines (reflective of primary and outgrowing metastatic tumor cells, respectively) require higher statin concentrations than mesenchymal-like tumor cells (reflective of in-circulation metastatic tumor cells) to achieve the same degree of growth inhibition. Here, we show that attenuation of HMGCR expression in the presence of atorvastatin leads to stronger growth inhibition than dual target blockade of the mevalonate pathway in relatively statin resistant cell lines, mainly through inhibition of protein prenylation pathways. Thus, combined inhibition of the mevalonate pathway's rate-limiting enzyme, HMGCR, can improve atorvastatin's growth inhibitory effect on epithelial- and mixed mesenchymal-epithelial cancer cells, a finding that may have implications for the design of future anti-metastatic cancer therapies.

Background: Breast cancers can recur after a long latency period following ‘successful’ primary treatments. Chronic inflammation significantly correlates with reduced diseased-free survival in breast cancer patients and could be a point of intervention to prevent recurrence. Liver is among the main sites of breast cancer recurrence. Thus, we hypothesise that inflammatory signals from hepatic stellate cells, the major inflammatory regulators in the sinusoid, could stimulate dormant cancer cells to emerge. Methods: We utilise in vitro co-culture of breast cancer cells with stellate cells and an ex vivo 3D human liver micro-physiologic system to identify stellate cells-derived factors that mediate tumour emergence. Results: Activated, but not quiescent, hepatic stellate cells secreted soluble factors to induce the proliferation of MCF7 and MDA-MB231 cancer cells. IL-8 and MCP-1 were highly secreted by the activated stellate cells and primary human non-parenchymal cells. IL-8 significantly reduced serum-starvation growth arrest on MDA-MB231 cells in vitro and increased cancer proliferation ex vivo. Blocking IL-8Rb/CXCR2 reduced IL-8-induced cancer growth and proliferation. Conclusions: Activated stellate cells can induce breast cancer emergence from dormancy in the liver by secreting inflammatory cytokines. Preventing liver inflammation or disrupting the subsequent key cytokines may prevent metastatic outgrowth.

BACKGROUND: Previous studies have had inconsistent findings regarding the quantity and frequency of prenatal alcohol exposure (PAE) that lead to deficits in growth and neurodevelopment. This may be due to imprecise methods of exposure classification. Our objective in this study was to employ longitudinal trajectory modeling of maternal drinking patterns associated with infant growth or neurodevelopmental deficits to a homogenous sample of mothers and infants. METHODS: From a sample of 471 pregnant women prospectively enrolled in a longitudinal study in the Ukraine, we performed a longitudinal cluster analysis of drinking patterns across gestation. We employed multivariable regression analyses to determine if each trajectory group was associated with infant weight, length, or head circumference at birth or psychomotor or mental deficits in infancy. RESULTS: We identified 5 distinct PAE trajectory groups: minimal or no PAE throughout gestation, low-to-moderate PAE with discontinuation early in gestation, low-to-moderate PAE sustained across gestation, moderate-to-high PAE with reduction early in gestation, and high PAE sustained across gestation. The highest-trajectory group was associated with deficits in infant weight and length at birth and deficits in psychomotor and mental performance at 6 to 12 months of age. Although confidence intervals overlapped, low-to-moderate sustained use was more strongly associated with most negative infant outcomes than moderate-to-high PAE with early reduction. CONCLUSIONS: With these findings, we confirm that high, sustained PAE confers the highest risk for adverse infant outcomes but demonstrate that even low-to-moderate PAE continued across gestation is associated with certain deficits. This approach may be used to help clinicians identify high-risk infants for targeted early intervention.

Even the most therapy responsive melanomas eventually evade immune checkpoint inhibition. Interestingly, initial responses seem to benefit from localized inflammation, but this can become prohibitive when treating advanced disease. Subsequently, there is an urgent need to elucidate how inflammation impacts the tumor microenvironment (TME). Here, inhibition of EGFR and Ras/Raf/MEK is shown to drive the release of a pro-inflammatory secretome that activates the TME to promote progression. This secretome was enriched with immune recruiting cytokines and members of the transforming growth factor beta (TGFβ) superfamily. Both healthy and cancer associated fibroblasts (CAFs) were hyperactivated in response to the inflamed secretome with induction of the inflammatory marker, alpha smooth muscle actin (αSMA). Pharmacologic inhibition of TGFβ receptor type I (TGFβRI) using SB431542 or clinically relevant TEW-7197, diminished CAF αSMA phenotype, disrupted TGFβ and phospo-SMAD2/3 signaling, and blunted CAF expression of tumor promoting extracellular matrix and adhesion molecules. Invasion of melanoma spheroids was enhanced ~2.5-fold in CAF coculture transwells. However, treatment with MEK inhibitor AZD6244 and TEW-7197 only limited invasion of mixed melanoma and CAF multi-cell spheroids in vitro. Prompted by this, the extracellular matrix of patient-derived and melanoma xenograft tumors was analyzed for inflammatory protein expression. Micrometastatic foci were flanked by peri-tumor αSMA+ CAFs and enriched for inflammatory Tenascin-C (TNC) at the invasive front. The analysis of human melanoma tissue microarrays corroborated the experimentally observed expression of αSMA CAFs and TNC which statistically increase with malignant tumor grade. Conversely, the anti-invasive Decorin (DCN), decreased in malignant melanoma while collagen-1 increased during late stage tumor fibrosis. These findings prompted the use of ex vivo human skin organ cultures (SOCs) to define how cell matrix impacted melanoma. Addition of DCN to the SOC eliminated melanoma vertical invasion whereas pathological TNC promoted progression. Thus, a BRAFV600E resistant patient-derived metastatic cell line was used to establish intrasplenic xenografts that were treated after one week using AZD6244 and TEW-7197. Both monotherapy and combination therapy were well tolerated and resulted in reduced splenic tumor burden, αSMA staining, and halted metastasis to the liver. Citation Format: Andrew M. Bradshaw, Erica Kuo, Jelena Grahovac, Kyle Sylakowski, Cindy Sander, Howard Edington, John M. Kirkwood, Alan Wells. The therapeutically inflamed tumor microenvironment drives melanoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3169.

How microenvironmental inflammation impacts melanoma progression remains controversial. Inflammation appears beneficial during interferon stimulated expression of checkpoint targets. However, when tipped too far, therapy-driven inflammation has been linked to debilitating autoimmune disease in cancer patients. One unanswered question is whether this inflammation also promotes melanoma dissemination. Here, drug-stressed melanomas are shown to promote progression via a pro-inflammatory secretome that activates the tumor microenvironment. Analyses showed the drug specific activation of immune recruiting cytokines and the transforming growth factor beta (TGFβ) superfamily. In coculture, TGFβ from drug-stressed melanomas resulted in the paracrine hyperactivation of cancer associated fibroblasts (CAFs) as noted by the inflammatory marker alpha smooth muscle actin (αSMA). Pharmacologic inhibition of TGFβ using SB431542 or clinically relevant EW-7197, blunted the αSMA phenotype as well as downstream phospho-SMAD activation. Invasion of melanoma spheroids in CAF cocultures resulted in enhanced invasion which could suppressed by targeted MEK inhibition (AZD6244) and TGFβ receptor blockade. Engrafted melanomas in vivo presented with the rapid expansion of peri-tumoral αSMA+ CAFs surrounding invasive micrometastatic nodules in mouse livers. We have previously reported that extracellular matrix (ECM) protein tenascin-C (TNC) is enriched at the invasive fronts of melanoma, which was recapitulated in vivo and correlated to human melanomas. Human tissue microarrays were assayed for pro-inflammatory TNC, healthy ECM marker Decorin (DCN), αSMA CAFs, and fibrotic Collagen (COL1A1) expression. We detected statistically significant increases in TNC, αSMA and COL1A1 correlating to progression and a concomitant decrease in DCN. Evidence from drug treated spheroid cocultures did not show complete suppression single cell invasion. Prompted by this, ex vivo organotypic skin organ cultures containing TNC and DCN were developed to show that the ECM provides physiological pro-and-anti invasive signals. Expression of DCN therapeutically inhibited collective and single cell invasion in this model. We also observed transcriptomic suppression of TNC and key inflamed matrisome proteins in anti-TGFβ treated CAFs, even when challenged with TGFβ conditioned media. Informed by this, we used a TNC+ vemurafenib resistant patient-derived metastatic melanoma in our xenograft model. Cohorts treated with MEK inhibitor and combination therapy with EW-7197 showed drug efficacy and tolerance with detectable decreases in αSMA and tumor burden of the spleen. Treated animals also showed reduced liver metastases compared to control. Taken together, combination therapy that blocks off-target inflammation and cancer proliferation represent an underappreciated but potentially successful strategy to halt melanoma progression. Citation Format: Andrew Bradshaw, Erica Kuo, Jelena Grahovac, Cindy Sander, Howard Edington, John M. Kirkwood, Alan Wells. The therapeutically inflamed tumor microenvironment drives melanoma progression [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT002.

BACKGROUND AND METHODS Prostate cancer frequently expresses an osteomimetic phenotype, but it is unclear how it is regulated and what biological and clinical implications it confers. Because mechanical forces physiologically regulate bone-remodeling activity in osteocytes, we hypothesized that mechanical action of fluid flow (MAFF) at the cancer microenvironment may similarly foster prostate cancer cell osteomimicry. RESULTS We showed that in vitro MAFF on androgen-dependent (LNCap) and androgen-independent (PC3) prostate cancer cells remarkably increased OPG, VEGF, RunX2, PTH1R, and PTHrP gene expression in both cell lines irrespective of their androgen dependency. MAFF also altered the cytokine secretion pattern of prostate cancer cells, including Ang2, SCF, and TNFα increase with TRAIL decrease in the supernatant of both cell lines; preferential increase of Leptin and PDGF-BB in LnCap and of VEGF, IL-8, and G-CSF in PC3; and exclusive increase of FGFβ, MIF, and PECAM-1 with HGF decrease in LnCap, and of TGBβ1, HGF, M-CSF, CXCL1, and CCL7 with NGF decrease in PC3. Murine MLO-Y4 osteocyte-conditioned medium (CM) abrogated M-CSF, G-CSG, IL-8, TNFα, and FGFβ secretion-stimulating activity of mechanical stimulation on PC3 cells, and did the opposite effect on LnCap cells. However, MAFF fostered osteomimetic gene expression response of PC3 cells, but not of LnCap cells, to mechanically stimulated osteocyte-CM. Moreover, it abrogated TNFα and IL-8 secretion inhibitory effect of osteocyte-CM on mechanically stimulated PC3 cells and G-CSF, TNFα, and FGFβ-stimulating effect on mechanically stimulated LnCap cells. CONCLUSIONS MAFF activated osteoblast-like phenotype of prostate cancer cells and altered their responses to osteocyte soluble factors. It also induced osteocyte production of osteomimetic gene expression- and cytokine secretion-stimulating factors for prostate cancer cells, particularly, when they were mechanically stimulated. Importantly, MAFF induced a prometastatic response in androgen-independent prostate cancer cells, suggesting the interest of mechanical stimulation-dependent transcription and secretion patterns as diagnostic biomarkers, and as therapeutic targets for the screening of bone-metastasizing phenotype inhibitors upregulated during prostate cancer cell response to MAFF at the cancer microenvironment. Prostate 77:321–333, 2017. © 2016 Wiley Periodicals, Inc.

The liver is a highly metastasis-permissive organ, tumor seeding of which usually portends mortality. Its unique and diverse architectural and cellular composition enable the liver to undertake numerous specialized functions, however, this distinctive biology, notably its hemodynamic features and unique microenvironment, renders the liver intrinsically hospitable to disseminated tumor cells. The particular focus for this perspective is the bidirectional interactions between the disseminated tumor cells and the unique resident cell populations of the liver; notably, parenchymal hepatocytes and non-parenchymal liver sinusoidal endothelial, Kupffer, and hepatic stellate cells. Understanding the early steps in the metastatic seeding, including the decision to undergo dormancy versus outgrowth, has been difficult to study in 2D culture systems and animals due to numerous limitations. In response, tissue-engineered biomimetic systems have emerged. At the cutting-edge of these developments are ex vivo ‘microphysiological systems’ (MPS) which are cellular constructs designed to faithfully recapitulate the structure and function of a human organ or organ regions on a milli- to micro-scale level and can be made all human to maintain species-specific interactions. Hepatic MPSs are particularly attractive for studying metastases as in addition to the liver being a main site of metastatic seeding, it is also the principal site of drug metabolism and therapy-limiting toxicities. Thus, using these hepatic MPSs will enable not only an enhanced understanding of the fundamental aspects of metastasis but also allow for therapeutic agents to be fully studied for efficacy while also monitoring pharmacologic aspects and predicting toxicities. The review discusses some of the hepatic MPS models currently available and although only one MPS has been validated to relevantly modeling metastasis, it is anticipated that the adaptation of the other hepatic models to include tumors will not be long in coming.

Pericytes have generally been considered in the context of stabilizing vessels, ensuring the blood barriers, and regulating the flow through capillaries. However, new reports suggest that pericytes may function at critical times to either drive healing with minimal scarring or, perversely, contribute to fibrosis and ongoing scar formation. Beneficially, pericytes probably drive much of the vascular involution that occurs during the transition from the regenerative to the resolution phases of healing. Pathologically, pericytes can assume a fibrotic phenotype and promote scarring. This perspective will discuss pericyte involvement in wound repair and the relationship pericytes form with the parenchymal cells of the skin. We will further evaluate the role pericytes may have in disease progression in relation to chronic wounds and fibrosis.

Breast cancer (BrCa) mortality continues to result predominately from distant metastases that can emerge years after successful treatment of the primary disease. Metastatic resistance to agents that eradicate the primary mass is likely due to protection from the metastatic microenvironment and the quiescent state of dormant BrCa cells. Advancements for the treatment of metastatic tumors have been made, but significant progress has been hampered by the lack of relevant model systems, particularly for dormancy. We address this gap with an innovative all-human 3D liver microphysiological system (MPS). The liver is both a major site for BrCa metastasis (and other solid tumors) and the primary site of drug metabolism and limiting toxicities, an important consideration in evaluating cancer therapy efficacy and availability. Primary hepatocytes and non-parenchymal cells (NPC) from human liver resections were seeded into the MPS. Following tissue formation on day 3, tagged BrCa cells were seeded and allowed a minimum of 4 days to integrate into the tissue before interventions were initiated. On day 7, chemotherapy treatment of micrometastases was initiated for 72h. Cultures were allowed 3 days to recover before the MPS was challenged with inflammatory factors (LPS/EGF) for 48h. BrCa cells were then re-treated with chemotherapy (either the same or alternate therapy) on day 21 for 72h. Hepatocyte function and injury were measured by urea, AST, ALT, A1AT, fibrinogen and CYP P450 assays. BC proliferation was monitored by quantification, Ki67 staining, and EdU incorporation. Communication networks within the metastatic microenvironment during different stages of metastatic BrCa progression were identified using Luminex assays (55 analytes). The metastatically aggressive MDA-MB-231 BrCa cells demonstrated growth attenuation after 12d of culture in a subpopulation of cells (Ki67-/EdU-). Treatment of BrCa cells with doxorubicin for 72h eradicates the cycling cells, leaving behind a dormant cell population (Ki67-/EdU-) that can be subsequently stimulated to cycle by addition of inflammatory stimuli. A second dose of doxorubicin or cisplatin reduced the BrCa load but did not eradicate the BrCa. Luminex analysis of culture supernatants identified signaling molecules potentially involved in metastatic progression. In addition, we present the use of adjuvant therapy in the MPS to prevent this outgrowth of the dormant tumor cells. In parallel, we have piloted hydrogel scaffolds that better support tissue formation and produce signals consistent with a healthier liver physiology. Hydrogels enhanced MDA-MB-231 cell entry into dormancy, resulting in reduced efficacy of doxorubicin treatment with greater persistence of tumor load. The MPS provides a mechanism to close the gap in understanding metastatic dormancy. We demonstrate spontaneous dormancy for the first time in an all-human system and mimicked the dormancy and outgrowth observed in patients. Namely, that dormant BrCa are resistant to chemotherapy and can be stimulated to reemerge following an inflammatory insult. The completion of these studies will provide insights into the tumor biology of metastatic seeding, dormancy, and re-emergence and provide an accessible tool for testing therapeutics against metastatic BrCa in a metabolically competent system capable of evaluating dose-limiting toxicity. Citation Format: Beckwitt C, Wheeler SE, Clark AM, Pillai VC, Young CL, Stolz DB, Lauffenburger DA, Venkataramanan R, Griffith LG, Wells A. Breast cancer dormancy, re-emergence, and treatment. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-19.

We thank Drs. Lin and Chuu for their recent insightful Commentary in TAU , on our paper in Hepatology concerning the role of E-cadherin in the survival and chemoresistance of prostate cancer metastases (1). We would like to take this opportunity to amplify or explain some of the findings as they discussed.

Purpose Mitomycin C is routinely applied during trabeculectomy surgeries to enhance bleb survival after glaucoma filtration surgery. The current approach involves placing cellulose sponges soaked in mitomycin C at a standard concentration onto bare sclera for a predetermined duration, which varies among surgeons. The purpose of this study was to compare the effects of sponge-applied versus intra-Tenon injection of mitomycin C during modified trabeculectomy. Methods Two groups of five New Zealand White rabbits underwent glaucoma filtration surgery with either preoperative intra-Tenon injection of mitomycin C or intraoperative application of mitomycin C using a cellulose sponge. Postoperative intraocular pressure was recorded weekly, and eyes were enucleated and sent for pathological examination and histological analysis. Results An intra-Tenon injection of mitomycin C resulted in decreased intraocular pressure measurements and bleb vascularity compared to the controls but increased levels compared to the sponge-applied group. Collagen deposition and cellularity were reduced and the goblet cell population was increased in the intra-Tenon injection group. Conclusions This study shows that an intra-Tenon injection can be an effective method for administering mitomycin C compared to the standard-of-care approach of mitomycin C being sponge applied onto bare sclera. Mitomycin C injection led to a greater reduction in intraocular pressure and inhibition of fibroblasts. The associated goblet cell population that can lead to increased mitomycin C toxicity-related morbidity was minimized with the intra-Tenon injection compared to the sponge-applied MMC treatment. Therefore, patients with ocular surface disease may benefit from an intra-Tenon injection. Translational relevance This project provides a direct, qualitative assessment in an animal model of common techniques within glaucoma filtration surgery for drug delivery to improve surgical success.

Epidemiologic studies have, variably, shown the concomitant use of statin drugs to be beneficial to cancer outcomes. Statin drugs have been FDA approved for three decades for the treatment of high cholesterol and atherosclerotic coronary artery disease and are widely used. This has engendered studies as to their influence on concomitant diseases, including cancers. In this context, statin use has been correlated, variably, with a decrease in deaths from breast cancer. However, there is no extant model for this effect, and the extent of efficacy is open to question. The overarching goal of this article is to communicate to the reader of the potential of statins to reduce breast cancer progression and mortality. This is the use as a secondary prevention measure, and not as a therapy to directly counter active cancer. First, salient aspects of statin pharmacology, as relates to cardiovascular disease, will be discussed. Second, the basic and clinical research studies that investigate statin usage in breast cancer will be presented. Additionally, statin effects in other cancer types will be included for context. Finally, proposals for future basic and clinical research studies to determine the role of statins in breast cancer management will be presented. Electronic supplementary material The online version of this article (10.1186/s13058-018-1066-z) contains supplementary material, which is available to authorized users.

Background Metastasis in breast cancer foreshadows mortality, as clinically evident disease is aggressive and generally chemoresistant. Disseminated breast cancer cells often enter a period of dormancy for years to decades before they emerge as detectable cancers. Harboring of these dormant cells is not individually predictable, and available information suggests that these micrometastatic foci cannot be effectively targeted by existing therapies. As such, long-term, relatively non-toxic interventions that prevent metastatic outgrowth would be an advance in treatment. Epidemiological studies have found that statins reduce breast cancer specific mortality but not the incidence of primary cancer. However, the means by which statins reduce mortality without affecting primary tumor development remains unclear. Methods We examine statin efficacy against two breast cancer cell lines in models of breast cancer metastasis: a 2D in vitro co-culture model of breast cancer cell interaction with the liver, a 3D ex vivo microphysiological system model of breast cancer metastasis, and two independent mouse models of spontaneous breast cancer metastasis to the lung and liver, respectively. Results We demonstrate that statins can directly affect the proliferation of breast cancer cells, specifically at the metastatic site. In a 2D co-culture model of breast cancer cell interaction with the liver, we demonstrate that atorvastatin can directly suppress proliferation of mesenchymal but not epithelial breast cancer cells. Further, in an ex vivo 3D liver microphysiological system of breast cancer metastasis, we found that atorvastatin can block stimulated emergence of dormant breast cancer cells. In two independent models of spontaneous breast cancer metastasis to the liver and to the lung, we find that statins significantly reduce proliferation of the metastatic but not primary tumor cells. Conclusions As statins can block metastatic tumor outgrowth, they should be considered for use as long-term adjuvant drugs to delay clinical emergence and decrease mortality in breast cancer patients.

Cytokines and chemokines are potent modulators of brain development and as such, dysregulation of the maternal immune system can result in deviations in the fetal cytokine balance, altering the course of typical brain development, and putting the individual on a "pathway to pathology". In the current study, we used a multi-variate approach to evaluate networks of interacting cytokines and investigated whether alterations in the maternal immune milieu could be linked to alcohol-related and alcohol-independent child neurodevelopmental delay. This was achieved through the measurement of 40 cytokines/chemokines from maternal blood samples collected during the second and third trimesters of pregnancy. Importantly, during the second trimester we identified network enrichment in levels of cytokines including IFN-ɣ, IL-10, TNF-β, TNF-α, and CRP associated with offspring neurodevelopmental delay. However, as elevations in levels of these cytokines have previously been reported in a wide range of neurodevelopmental disorders including autism spectrum disorder and schizophrenia, we suggest that this cytokine profile is likely not disorder specific, but rather may be an indicator of neurodevelopmental delay in general. By contrast, distinct clusters of activated/inhibited cytokines were identified based on maternal alcohol consumption and child neurodevelopmental outcome. Specifically, cytokines including IL-15, IL-10, MDC, and members of the VEGF sub-family were highest in alcohol-consuming mothers of children with neurodevelopmental delay and were identified in both network analyses and examination of individual cytokines, whereas a differential and unique cytokine profile was identified in the case of alcohol-independent child neurodevelopmental delay. We propose that the current findings could provide a critical step towards the development of early biomarkers and possibly interventions for alcohol-related neurodevelopmental delay. Importantly, the current approach could be informative for understanding mechanisms linking maternal immune system dysfunction and adverse child outcomes in a range of other neurodevelopmental disorders.

Most breast cancer (BrCa) mortality results from distant metastases. Current evidence strongly suggests that in some instances these disseminated cells remain dormant for long periods of time. Both the non-proliferative state and protective microenvironment of the metastatic niche likely contribute to the observed resistance of metastases to chemotherapies that are otherwise effective against the primary tumor. Although significant interventional progress has been made on primary tumors, the lack of relevant accessible model systems for metastases has hindered the development of therapies against this stage. To address this gap, we developed an innovative all-human 3D ex vivo hepatic microphysiological system (MPS) to faithfully reproduce human physiology and thereby facilitate the investigation of BrCa behavior in a micrometastatic niche. The liver is a major site of metastasis for carcinomas and is also the primary site of drug metabolism (activation and/or detoxification), which is a significant factor in determining efficacy and limiting toxicities of cancer therapies. The MPS incorporates hepatocytes and nonparenchymal cells (NPC) isolated from fresh human liver resections. BrCa cells (RFP+) are seeded on day 3 and afforded time to intercalate into the hepatic tissue until treatment with chemotherapy on day 7 for 72h. Surviving BrCa cells are stimulated on day 13 with LPS/EGF and cultured through day 15. Proliferation is monitored by RFP quantification, Ki67 staining and EdU incorporation. Physiological function of the hepatic tissue is monitored throughout the experiment by protein catabolism (urea), active metabolism (glucose, CYP P450) and injury markers (AST, ALT, A1AT, fibrinogen). Luminex assays (55 analytes) were used to provide insights into the communication networks in the hepatic metastatic milieu during different stages of dormancy and progression, and identify potential metastatic biomarkers via computational approaches. The MPS maintains the physiologic function of the hepatic niche through 15 days and BrCa cells effectively integrate into the established niche. Spontaneous dormancy is observed amongst a subpopulation of BrCa cells, indicated by the absence of Ki67 staining and EdU incorporation after 12 days of culture. Further, we demonstrate that the BrCa cells surviving chemotherapy (doxorubicin) are non-proliferating (Ki67-/EdU-). Notably, ‘re-awakening’ of the surviving non-proliferating cancer cells is observed in the presence of physiological inflammatory stressors (LPS/EGF). Luminex analyses of the milieu effluent identified signaling molecules from NPC influenced the metastatic cell fraction entering dormancy. This MPS provides unprecedented insights into the tumor biology of dormant micrometastases. We demonstrate the recreation of spontaneous, rather than engineered, BrCa dormancy in an all-human ex vivo hepatic MPS. Mimicking the dormancy and outgrowth observed in patients, we found that dormant breast cancer cells that are resistant to chemotherapy can be stimulated to re-emerge following an inflammatory insult. Ultimately, this MPS provides an accessible tool to identify new therapeutic strategies for metastasis during initial seeding, dormancy and re-emergence, while concurrently evaluating agent efficacy for metastasis, metabolism and dose-limiting toxicity. Citation Format: Amanda M Clark, Sarah E Wheeler, Donald P Taylor, Carissa L Young, Venkateswaran C Pillai, Donna B Stolz, Raman Venkataramanan, Douglas A Lauffenburger, Linda G Griffith, Alan Wells. Modeling breast cancer dormancy and re-emergence [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-01.

The HMG-CoA reductase inhibitors, statins, have been used as lipid lowering drugs for decades and several epidemiological studies suggest statin usage correlates with a decreased incidence of cancer specific mortality in patients. However, the mechanism of this mortality benefit remains unclear. Here, we demonstrate that statin drug lipophilicity and affinity for its target enzyme, HMGCR, determine their growth suppressive potency against various tumor cell lines. The lipophilic atorvastatin decreases cancer cell proliferation and survival in vitro. Statin sensitivity coincided with Ras localization to the cytosol instead of the membrane, consistent with a decrement in prenylation. To investigate signaling pathways that may be involved with sensitivity to statin therapy, we employed inhibitors of the PI3K-Akt and Mek-Erk signaling cascades. We found that inhibition of PI3K signaling through Akt potentiated statin sensitivity of breast cancer cells in vitro and in co-culture with primary human hepatocytes. The same effect was not observed with inhibition of Mek signaling through Erk. Moreover, the sensitivity of breast cancer cells to atorvastatin-mediated growth suppression correlated with a decrease in EGF-mediated phosphorylation of Akt. As an increase in Akt activity has been shown to be involved in the metastasis and metastatic outgrowth of many cancer types (including breast), these data suggest a mechanism by which statins may reduce cancer specific mortality in patients.

Objective By the 1990s it became popular for women to use hormone therapy (HT) to ease menopause symptoms. Bioidentical estrogen and progesterone are supplements whose molecular structures are identical to what is made in the human body, while synthetic supplements are ones whose structures are not. After the Women's Health Initiative found that the combined use of the synthetics conjugated equine estrogen (CEE) and medroxyprogesterone acetate (MPA) increased breast cancer risk, prescriptions for synthetic HT declined considerably. Since then there has been an increased interest in bioidentical HT; today there are a plethora of websites touting their benefits. However, no peer-reviewed articles support these claims. We performed a retrospective study with the objective of verifying the hypothesis that bioidentical HT is associated with decreased breast cancer risk than CEE & MPA. Methods We searched The Northwestern Medicine Enterprise Data Warehouse for women who initiated HT use after age 50. Women who did not take any HT drug after age 50 served as controls. Nine HT protocols were investigated for breast cancer risk. Results Significant results include CEE Alone is associated with decreased breast cancer risk (HR = 0.31), Other Synthetic Estrogen Alone is associated with increased breast cancer risk (HR = 1.49), Bioidentical Estrogen Alone is associated with decreased breast cancer risk(HR = 0.65), CEE & MPA is associated with reduced breast cancer risk (HR = 0.43), and CEE & MPA is associated with reduced breast cancer risk relative to Bioidentical Estrogen & Progesterone (HR = 0.25). Discussion Our results indicate CEE & MPA is superior to bioidentical HT as far as breast cancer risk. Furthermore, this combination is associated with decrease of breast cancer risk, contrary to previous findings. Additional retrospective studies are needed to confirm our results.

Breast cancer mortality predominantly results from dormant micrometastases that emerge as fatal outgrowths years after initial diagnosis. In order to gain insights concerning factors associated with emergence of liver metastases, we recreated spontaneous dormancy in an all-human ex vivo hepatic microphysiological system (MPS). Seeding this MPS with small numbers (

Severe hepatic inflammation is a common cause of acute liver injury following systemic infection with Ehrlichia, obligate Gram-negative intracellular bacteria that lack lipopolysaccharide (LPS). We have previously shown that type I IFN (IFN-I) and inflammasome activation are key host-pathogenic mediators that promote excessive inflammation and liver damage following fatal Ehrlichia infection. However, the underlying signals and mechanisms that regulate protective immunity and immunopathology during Ehrlichia infection are not well understood. To address this issue, we compared susceptibility to lethal Ixodes ovatus Ehrlichia (IOE) infection between wild type (WT) and MyD88-deficient (MyD88-/-) mice. We show here that MyD88-/- mice exhibited decreased inflammasome activation, attenuated liver injury, and were more resistant to lethal infection than WT mice, despite suppressed protective immunity and increased bacterial burden in the liver. MyD88-dependent inflammasome activation was also dependent on activation of the metabolic checkpoint kinase mammalian target of rapamycin complex 1 (mTORC1), inhibition of autophagic flux, and defective mitophagy in macrophages. Blocking mTORC1 signaling in infected WT mice and primary macrophages enhanced bacterial replication and attenuated inflammasome activation, suggesting autophagy promotes bacterial replication while inhibiting inflammasome activation. Finally, our data suggest TLR9 and IFN-I are upstream signaling mechanisms triggering MyD88-mediated mTORC1 and inflammasome activation in macrophages following Ehrlichia infection. This study reveals that Ehrlichia-induced liver injury and toxic shock are mediated by MyD88-dependent inflammasome activation and autophagy inhibition., Author summary Human monocytic ehrlichiosis (HME) is the most prevalent emerging infectious disease in the United States. Ehrlichia chaffeensis, etiologic agent of HME, is a Gram negative obligate intracellular bacterium transmitted by infected tick bites and can infect different cell type. Although Ehrlichia lack lipopolysaccharide (LPS), they induce potentially life threatening HME that mimic sepsis or toxic shock associated with multi-organ failure. The clinical diagnosis of HME is difficult, and definitive diagnosis is most often retrospective. Late antibiotic treatment is frequently ineffective in preventing disease progression to fatal multi-organ failure. Liver failure is one of the most serious complications and the most frequent cause of death in patients with HME, however we only have a limited understanding of how this liver failure is caused during fatal Ehrlichia infection. The objective of this study is to determine how LPS-negative Ehrlichia activates inflammatory responses in macrophages during Ehrlichia infection to promote liver damage. We show here that MyD88-signaling causes detrimental derangement of the immune system and subsequent liver damage by regulating two key innate immune events in macrophages: autophagy and inflammasome activation. Targeting host-pathogenic pathways in ehrlichiosis can be incorporated into future design of novel therapeutic approaches for HME.

Metastasis accounts for most cancer-related deaths. The majority of solid cancers, including those of the breast, colorectum, prostate and skin, metastasize at significant levels to the liver due to its hemodynamic as well as tumor permissive microenvironmental properties. As this occurs prior to detection and treatment of the primary tumor, we need to target liver metastases to improve patients' outcomes. Animal models, while proven to be useful in mechanistic studies, do not represent the heterogeneity of human population especially in drug metabolism lack proper human cell-cell interactions, and this gap between animals and humans results in costly and inefficient drug discovery. This underscores the need to accurately model the human liver for disease studies and drug development. Further, the occurrence of liver metastases is influenced by the primary tumor type, sex and race; thus, modeling these specific settings will facilitate the development of personalized/targeted medicine for each specific group. We have adapted such all-human 3D ex vivo hepatic microphysiological system (MPS) (a.k.a. liver-on-a-chip) to investigate human micrometastases. This review focuses on the sources of liver resident cells, especially the iPS cell-derived hepatocytes, and examines some of the advantages and disadvantages of these sources. In addition, this review also examines other potential challenges and limitations in modeling human liver.

α-Actinin-4 (ACTN4), a key regulator of the actin cytoskeleton, is up-regulated in melanoma, though its role in melanoma remains speculative. We have discovered that in WM1158, a highly aggressive melanoma cell line, down-regulation of ACTN4 by shRNA induces a collagen I-dependent amoeboidal-to-mesenchymal transition. Re-expression of low levels of WT ACTN4 but not similar expression levels of ACTN1 successfully restores the amoeboidal morphology and limits collagen I gel compaction. A truncated ACTN4 mutant 1-890, which lacks the C-terminal tail, fails to rescue the amoeboidal morphology and to compact collagen I gel. Interestingly, in three-dimensional collagen I gels, ACTN4 KD cells are more polarized compared with cells in which scrambled shRNA is expressed. Surprisingly, ACTN4 KD cells migrate faster than the ones expressing the scrambled shRNA on a collagen I gel (two-dimensional) although these two cell lines migrate similarly on tissue culture. Most importantly, down-regulation of ACTN4 significantly reduced invasion of WM1158 cells into the three-dimensional collagen I gel, a representative of the dermis. Taken together, these findings suggest that ACTN4 plays an important role in maintaining the amoeboidal morphology of invasive melanoma and thus promoting dissemination through collagen-rich matrices.

Treatment of triple negative breast cancer (TNBC) is particularly difficult due to lack of molecular targets. The vast majority of breast cancer patient mortality is a result of emergence of disseminated cancer cells from a so-called dormancy-like state allowing subsequent aggressive growth at the metastatic sites. Actin assembly and important regulators of actin assembly play a key role in promoting the dormancy to proliferation switch of extravasated breast cancer cells. MRTF (myocardin-related transcription factor - an important family of transcription cofactors for SRF)/SRF transcriptional axis plays a major role in transcriptional regulation of many major molecular components of actin cytoskeletal system. We hypothesize that MRTF plays a critical role in post-extravasation survival and outgrowth of TNBC cells. To test this hypothesis, we first performed matrigel-on-top assay (3D culture experiments which successfully predicts the post-extravasation pulmonary metastatic outgrowth competency of breast cancer cells) and found that loss-of-function (LOF) of MRTF, either by knockdown or small molecule inhibitor CCG-1423 (and its analog CCG-203971), significantly retards the outgrowth of isolated TNBC cells. In this model, LOF of MRTF also dramatically suppresses the progression of established outgrowth (an in vitro mimic of micro-to-macrometastasis progression) of TNBC cells. Translating these findings in vivo, in intracardiac-injection model of experimental metastasis, administration of MRTF inhibitor reduces the metastatic burden of TNBC cells in immunocompetent mice. These experimental data are consistent with clinical findings demonstrating significantly shorter progression-free survival with MRTF upregulation in breast cancer patients. To obtain further insight into the mechanism behind MRTF’s regulation of metastatic outgrowth, we performed RNAseq from TNBC grown in 3D culture treated with CCG and identified a number of differentially expressed genes related to cell proliferation/survival and invasion. In addition to these findings, we also found that LOF of MRTF causes a dramatic reduction of intracellular content of actin-binding protein Profilin-1 (Pfn1), an important regulator of actin cytoskeletal dynamics. Using a tetracycline-inducible Pfn1 knockdown system, we further demonstrated that acute Pfn1 depletion alone is sufficient to cause a major reduction of metastatic colonization of TNBC cells in vivo. Collectively, these findings suggest that MRTF-Pfn1 is an important signaling axis for metastatic outgrowth of TNBC cells. Citation Format: David M. Gau, Souvik Chakraborty, David Boone, Alan Wells, Partha Roy. MRTF|Profilin is an important signaling axis for metastatic outgrowth of triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-043.

ObjectiveA Clinical Decision Support System (CDSS) that can amass Electronic Health Record (EHR) and other patient data holds promise to provide accurate classification and guide treatment choices. Our objective is to develop the Decision Support System for Making Personalized Assessments and Recommendations Concerning Breast Cancer Patients (DPAC), which is a CDSS learned from data that recommends the optimal treatment decisions based on a patient's features.MethodWe developed a Bayesian network architecture called Causal Modeling with Internal Layers (CAMIL), and an algorithm called Treatment Feature Interactions (TFI), which learns from data the interactions needed in a CAMIL model. Using the TFI algorithm, we learned interactions for six treatments from the LSDS-5YDM dataset. We created a CAMIL model using these interactions, resulting in a DPAC which recommends treatments towards preventing 5-year breast cancer metastasis.ResultsIn a 5-fold cross-validation analysis, we compared the probability of being metastasis free in 5 years for patients who made decisions recommended by DPAC to those who did not. These probabilities are (the probability for those making the decisions appears first): chemotherapy (.938, .872); breast/chest wall radiation (.939, .902); nodal field radiation (.940, .784); antihormone (.941, .906); HER2 inhibitors (.934, .880); neadjuvant therapy (.931, .837). In an application of DPAC to the independent METABRIC dataset, the probabilities for chemotherapy were (.845, .788).DiscussionPatients who took the advice of DPAC had, as a group, notably better outcomes than those who did not. We conclude that DPAC is effective at amassing and analyzing data towards treatment recommendations. Some of the findings in DPAC are controversial. For example, DPAC says that chemotherapy increases the chances of metastasis for many node negative patients. This controversy shows the importance of developing a conclusive version of DPAC to ensure we provide patients with the best patient-specific treatment recommendations.

Cancer invasion is a complex process requiring, among other events, extensive remodeling of the extracellular matrix including deposition of pro-migratory and pro-proliferative moieties. In recent years, it has been described that while invading through matrices cancer cells can change shape and adapt their migration strategies depending on the microenvironmental context. Although intracellular signaling pathways governing the mesenchymal to amoeboid migration shift and vice versa have been mostly elucidated, the extracellular signals promoting these shifts are largely unknown. In this review, we summarize findings that point to matrikines that bind specifically to the EGF receptor as matricellular molecules that enable cancer cell migrational plasticity and promote invasion.

Tenascin-C (TNC), overexpressed in invasive growths, has been implicated in progression of melanoma, but the source and function of this molecule are not well defined. We found TNC expression at the front of invading melanoma cells, and that adding TNC to matrices enhances individual melanoma cell migration. As TNC is a multidomain protein, we examined the role of the TNC EGF-like (EGFL) repeats as these activate motogenic signaling cascades. We overexpressed a TNC fragment containing the assembly and EGFL domains of TNC (TNCEGFL). TNCEGFL-expressing melanoma cells had lower speed and persistence in 2D migration assays due to a shift in the adhesion-contractility balance, as expression of TNCEGFL delayed melanoma cell attachment and spreading. The less adhesive phenotype was due, in part, to increased Rho-associated kinase (ROCK) signaling concomitant with myosin light chain 2 and MYPT phosphorylation. Inhibition of ROCK activity, which drives transcellular contractility, restored adhesion of TNCEGFL-expressing melanoma cells and increased their migration in 2D. In contrast to the diminished migration in 2D, TNCEGFL-expressing melanoma cells had higher invasive potential in Matrigel invasion assays, with cells expressing TNCEGFL having amoeboid morphology. Our findings suggest that melanoma-derived TNCEGFL exert a role in melanoma invasion by modulating ROCK signaling and cell migration.

Distant metastasis is the major cause of breast cancer-related mortality, commonly emerging clinically after 5 or more years of seeming 'cure' of the primary tumor, indicating a quiescent dormancy. The lack of relevant accessible model systems for metastasis that recreate this latent stage has hindered our understanding of the molecular basis and the development of therapies against these lethal outgrowths. We previously reported on the development of an all-human 3D ex vivo hepatic microphysiological system that reproduces several features of liver physiology and enables spontaneous dormancy in a subpopulation of breast cancer cells. However, we observed that the dormant cells were localized primarily within the 3D tissue, while the proliferative cells were in contact with the polystyrene scaffold. As matrix stiffness is known to drive inflammatory and malignant behaviors, we explored the occurrence of spontaneous tumor dormancy and inflammatory phenotype. The microphysiological system was retrofitted with PEGDa-SynKRGD hydrogel scaffolding, which is softer and differs in the interface with the tissue. The microphysiological system incorporated donor-matched primary human hepatocytes and non-parenchymal cells (NPCs), with MDA-MB-231 breast cancer cells. Hepatic tissue in hydrogel scaffolds secreted lower levels of pro-inflammatory analytes, and was more responsive to inflammatory stimuli. The proportion of tumor cells entering dormancy was markedly increased in the hydrogel-supported tissue compared to polystyrene. Interestingly, an unexpected differential response of dormant cells to varying chemotherapeutic doses was identified, which if reflective of patient pathophysiology, has important implications for patient dosing regimens. These findings highlight the metastatic microphysiological system fitted with hydrogel scaffolds as a critical tool in the assessment and development of therapeutic strategies to target dormant metastatic breast cancer.

Liver is one of the most common sites of cancer metastasis. Once disseminated, the prognosis is poor as these tumors often display generalized chemoresistance, particularly for carcinomas that derive not from the aerodigestive tract. When these cancers seed the liver, the aggressive cells usually undergo a mesenchymal to epithelial reverting transition that both aids colonization and renders the tumor cells chemoresistant. In vitro studies demonstrate that hepatocytes drive this phenotypic shift. However, the in vivo evidence and the molecular signals that protect these cells from induced death are yet to be defined. Herein, we report that membrane surface E-cadherin-expressing prostate cancer cells were resistant to cell death by chemotherapeutic drugs but E-cadherin null cells or those expressing E-cadherin only in the cytoplasm were sensitive to death signals and chemotherapies both in vitro and in vivo. While cell-cell E-cadherin ligandation reduced mitogenesis, this chemoprotection was proliferation-independent as killing of both 5-ethynyl-2′-deoxyuridine-positive (or Ki67+) and 5-ethynyl-2′-deoxyuridine-negative (Ki67-) cells was inversely related to membrane-bound E-cadherin. Inhibiting the canonical survival kinases extracellular signal-regulated protein kinases, protein kinase B, and Janus kinase, which are activated by chemotherapeutics in epithelial cell-transitioned prostate cancer, abrogated the chemoresistance both in cell culture and in animal models of metastatic cancer. For disseminated tumors, protein kinase B disruption in itself had no effect on tumor survival but was synergistic with chemotherapy, leading to increased killing. Conclusion: Liver microenvironment-driven phenotypic switching of carcinoma cells and subsequent survival signaling results in activation of canonical survival pathways that protect the disseminated prostate cancer liver micrometastases in a proliferation-independent manner, and these pathways can be targeted as an adjuvant treatment to improve the efficacy of traditional chemotherapeutics (Hepatology 2016;64:1725-1742)

BACKGROUND & AIMS: Focal zone 1 steatosis, although rare in adults with nonalcoholic fatty liver disease (NAFLD), does occur in children with NAFLD. We investigated whether focal zone 1 steatosis and focal zone 3 steatosis are distinct subphenotypes of pediatric NAFLD. We aimed to determine associations between the zonality of steatosis and demographic, clinical, and histologic features in children with NAFLD. METHODS: We performed a cross-sectional study of baseline data from 813 children (age less than 18 years; mean age, 12.8±2.7 years). The subjects had biopsy-proven NAFLD and were enrolled in the NASH Clinical Research Network. Liver histology was reviewed using the Nonalcoholic Steatohepatitis Clinical Research Network scoring system. RESULTS: Zone 1 steatosis was present in 18% of children with NAFLD (n=146) and zone 3 steatosis was present in 32% (n=244). Children with zone 1 steatosis were significantly younger (10 vs 14 years; P