Tumour stemness and poor clinical outcomes in haemochromatosis patients with hepatocellular carcinoma.

AIMS: Patients with haemochromatosis (HFE) are known to have an increased risk of developing hepatocellular carcinoma (HCC). Available data are conflicting on whether such patients have poorer prognosis, and there is lack of data regarding the biology of HFE-HCC. We compared the course of HFE-HCC with a matched non-HFE-HCC control group and examined tumour characteristics using immunohistochemistry. METHODS: In this tertiary care-based retrospective analysis, 12 patients with HFE and 34 patients with alcohol/non-alcoholic steatohepatitis who underwent initially successful curative HCC therapy with ablation or resection were identified from our registry. Time to tumour progression was compared. Resected liver tissue from a separate cohort of 11 matched patients with HFE-HCC and without HFE-HCC was assessed for the expression of progenitor and epithelial-mesenchymal transition markers using immunohistochemistry. RESULTS: The median follow-up was 24.39 and 24.28 months for patients with HFE-HCC and those without HFE-HCC, respectively (p>0.05). The mean time to progression was shorter in the HFE group compared with the non-HFE group (12.87 months vs 17.78 months; HR 3.322, p<0.05). Patients with HFE-HCC also progressed to more advanced disease by the end of follow-up (p<0.05). Immunohistochemical analysis of matched HFE-HCC and non-HFE-HCC explants demonstrated increased expression of the cancer stem cell markers EpCAM (epithelial cell adhesion molecule) and EpCAM/SALL4 (spalt-like transcription factor 4) coexpression in HFE-HCC specimens (p<0.05). There was a high frequency of combined tumour subtypes within the HFE cohort. CONCLUSIONS: This study demonstrates that the clinical course of patients with HFE-HCC is more aggressive and provides the first data indicating that their tumours have increased expression of progenitor markers. These findings suggest patients with HFE-HCC may need to be considered for transplant at an earlier stage.

Implementing the Community Healthcare Network Model in Mid West community health care.

BACKGROUND: Integrated care, underpinned by a health and wellbeing approach, is central to Ireland's health service reform. This new Community Healthcare Network (CHN) model is in the process of implementation across Ireland as part of the Enhanced Community Care (ECC) Programme, a key deliverable of the Sláintecare Reform Programme that aims to 'shift left', ie change the way health care is delivered and bring more support closer to home. ECC aims to deliver integrated person-centred care, enhance Multidisciplinary Team (MDT) working, strengthen links with GPs and strengthen community supports. There are nine learning sites and 87 further CHNs.DeliverablesA new Operating ModelStrengthening governance and enhancing local decision-making through the development of a Community health network operating model, including a Community Healthcare Network Manager (CHNM), a GP Lead and Multidisciplinary Network Management Team.Enhancing primary care resources.Enhanced MDT workingProactive management of people with complex care needs in the community facilitated by the Multidisciplinary Team and new Clinical Coordinator (CC) and Key Worker (KW) roles.Redesign of the Clinical Team Meeting to allow virtual attendance and focused case discussion will facilitate GP attendance.Developing integrated care pathways between CHNs, Specialist Hubs (Chronic Disease and Frail Older Persons) and Acute Hospitals.Strengthening Community Supports, eg ALONE.Population Health ApproachPopulation health needs assessment utilising census data and health intelligence, local knowledge from GPs, PCTs, community services and service user engagement.Risk Stratification - resources applied intensively in a targeted manner to a defined population.Enhanced Health Promotion - addition of a Health Promotion and improvement officer to each CHN and the Healthy Communities Initiative, which aims to implement targeted initiatives to tackle challenges within specific communities, eg smoking cessation, social prescribing.Key enablers for implementationAppointment of a GP lead in all CHNs is essential to strengthen relationships and bring GP voice to health service reform.The CHN model has the potential to support the delivery of integrated care, providing opportunities for enhanced MDT working by identifying key personnel (CC, KW and GP lead) to support effective MDT functioning.Redesign of the clinical team meetings will support GP involvement and enhance collective decision making and joint working.Population risk stratification is necessary to deliver targeted services. CHNs need to be supported to carry out risk stratification. Furthermore, this is not possible without strong links with our CHN GPs and data integration.An integrated community case management system that can 'talk' to GP systems is a critical enabler for integration. EVALUATION: The Centre for Effective Services completed an early implementation evaluation of the 9 learning sites. From initial findings, it was concluded that there is an appetite for change, particularly in enhanced MDT working. Key features of the model, such as the introduction of the GP lead, clinical coordinators and population profiling, were viewed positively. However, respondents perceived communication and the change management process as challenging.

An integrative review of community nurse-led interventions to identify and respond to domestic abuse in the postnatal period.

AIM: To critically review community nurse-led domestic abuse interventions aimed at identifying and responding to domestic abuse in the postnatal period. BACKGROUND: Domestic abuse is a global problem resulting in dire consequences for women and children. Public Health Nurses (PHNs) are ideally placed to give women the opportunity to disclose in a safe and confidential manner; however, community settings present complex challenges. DESIGN: An integrative review and narrative summary. DATA SOURCES: Five electronic databases: CINAHL, MEDLINE, PsycINFO, EMBASE and Scopus, and peer-reviewed journals were searched for research papers published between 01 January 2005 and 01 March 2019. Fifteen papers met the inclusion criteria. REVIEW METHODS: An integrative review where qualitative and quantitative data were extracted. Following quality appraisal, data were collated, analysed and themes were identified. RESULTS: Quantitative outcomes from short-term interventions include an increase in routine enquiry, documentation of alone status and safety planning, however, referrals remained low. There was a reduction in victimization seen in intensive home visiting interventions. One study reported potential harm to mothers experiencing domestic abuse prior to the intervention. Thematic analysis generated three themes: (1) benefits to women and nurses, (2) approaches to domestic abuse identification and response and (3) implementation of community nurse-led interventions. CONCLUSION: Community nurse-led domestic abuse interventions have shown to have positive outcomes for women, provided the appropriate supports are in place such as: interagency training; guidelines, referral pathways and safety protocols; collaborative working with domestic abuse services and organizational support. IMPACT: Professionals such as PHNs are challenged to respond appropriately and compassionately to domestic abuse disclosures, while ensuring the safety of women and children is central to service delivery. This integrative review will inform further development, implementation and the sustainability of community nurse-led domestic abuse initiatives worldwide.

Comparison of clinical prediction rules for ruling out cirrhosis in nonalcoholic fatty liver disease (NAFLD).

BACKGROUND AND AIMS: Patients with nonalcoholic fatty liver disease (NAFLD) cirrhosis benefit from referral to subspecialty care. While several clinical prediction rules exist to identify advanced fibrosis, the cutoff for excluding cirrhosis due to NAFLD is unclear. This analysis compared clinical prediction rules for excluding biopsy-proven cirrhosis in NAFLD. METHODS: Adult patients were enrolled in the NASH Clinical Research Network (US) and the Newcastle Cohort (UK). Clinical and laboratory data were collected at enrolment, and a liver biopsy was taken within 1 year of enrolment. Optimal cutoffs for each score (eg, FIB-4) to exclude cirrhosis were derived from the US cohort, and sensitivity, specificity, positive predictive value, negative predictive value and AUROC were calculated. The cutoffs were evaluated in the UK cohort. RESULTS: 147/1483 (10%) patients in the US cohort had cirrhosis. All prediction rules had similarly high NPV (0.95-0.97). FIB-4 and NAFLD fibrosis scores were the most accurate in characterising patients as having cirrhosis (AUROC 0.84-0.86). 59/494 (12%) patients in the UK cohort had cirrhosis. Prediction rules had high NPV (0.92-0.96), and FIB-4 and NAFLD fibrosis score the most accurate in the prediction of cirrhosis in the UK cohort (AUROC 0.87-0.89). CONCLUSIONS: This cross-sectional analysis of large, multicentre international datasets shows that current clinical prediction rules perform well in excluding cirrhosis with appropriately chosen cutoffs. These clinical prediction rules can be used in primary care to identify patients, particularly those who are white, female, and <65, unlikely to have cirrhosis so higher-risk patients maintain access to specialty care.

Factors associated with engagement and adherence to a low-energy diet to promote 10% weight loss in patients with clinically significant non-alcoholic fatty liver disease.

OBJECTIVE: Clinical guidelines recommend weight loss to manage non-alcoholic fatty liver disease (NAFLD). However, the majority of patients find weight loss a significant challenge. We identified factors associated with engagement and adherence to a low-energy diet (LED) as a treatment option for NAFLD. DESIGN: 23 patients with NAFLD enrolled in a LED (~800 kcal/day) were individually interviewed. Transcripts were thematically analysed. RESULTS: 14/23 patients achieved ≥10% weight loss, 18/23 achieved ≥7% weight loss and 19/23 achieved ≥5% weight loss. Six themes were generated from the data. A desire to achieve rapid weight loss to improve liver health and prevent disease progression was the most salient facilitator to engagement. Early and significant weight loss, accountability to clinicians and regular appointments with personalised feedback were facilitators to engagement and adherence. The desire to receive positive reinforcement from a consultant was a frequently reported facilitator to adherence. Practical and emotional support from friends and family members was critically important outside of the clinical setting. Irregular working patterns preventing attendance at appointments was a barrier to adherence and completion of the intervention. CONCLUSIONS: Engagement and adherence to a LED in patients with NAFLD were encouraged by early and rapid weight loss, personalised feedback and positive reinforcement in the clinical setting combined with ongoing support from friends and family members. Findings support those identified in patients who completed a LED to achieve type 2 diabetes remission and highlight the importance of behaviour change support during the early stages of a LED to promote adherence.

Feasibility of a Very Low Calorie Diet to Achieve a Sustainable 10% Weight Loss in Patients With Nonalcoholic Fatty Liver Disease.

INTRODUCTION: Nonalcoholic fatty liver disease (NAFLD) is the most common liver condition worldwide. A weight loss goal of ≥10% is the recommended treatment for NAFLD; however, only a minority of patients achieve this level of weight reduction with standard dietary approaches. This study aimed to determine whether a very low calorie diet (VLCD) is an acceptable and feasible therapy to achieve and maintain a ≥10% weight loss in patients with clinically significant NAFLD. METHODS: Patients with clinically significant NAFLD were recruited to a VLCD (∼800 kcal/d) intervention using meal replacement products. Anthropometrics, blood tests (liver and metabolic), liver stiffness, and cardiovascular disease risk were measured at baseline, post-VLCD, and at 9-month follow-up. RESULTS: A total of 45 patients were approached of which 30 were enrolled 27 (90%) completed the VLCD intervention, and 20 (67%) were retained at 9-month follow-up. The VLCD was acceptable to patients and feasible to deliver. Intention-to-treat analysis found that 34% of patients achieved and sustained ≥10% weight loss, 51% achieved ≥7% weight loss, and 68% achieved ≥5% weight loss at 9-month follow-up. For those completing the VLCD, liver health (liver enzymes and liver stiffness), cardiovascular disease risk (blood pressure and QRISK2), metabolic health (fasting glucose, HbA1c, and insulin), and body composition significantly improved post-VLCD and was maintained at 9 months. DISCUSSION: VLCD offers a feasible treatment option for some patients with NAFLD to enable a sustainable ≥10%, weight loss, which can improve liver health, cardiovascular risk, and quality of life in those completing the intervention.

Performance of the PRO-C3 collagen neo-epitope biomarker in non-alcoholic fatty liver disease.

There is an unmet need for non-invasive biomarkers in non-alcoholic fatty liver disease (NAFLD) that can diagnose advanced disease and identify patients suitable for clinical trials. The PRO-C3 collagen neo-epitope is a putative direct marker of fibrogenesis. We assessed the performance of PRO-C3 in a large, well-characterised international NAFLD cohort and report the development and validation of 2 novel panels for the diagnosis of advanced fibrosis (F≥3) in NAFLD, including a simplified clinical score which eliminates the need for online calculators. METHODS: Plasma PRO-C3 levels were determined in a prospectively recruited international cohort of 449 patients with biopsy diagnosed NAFLD across the full disease spectrum (F0: n = 90; F1: 100; F2: 92; F3: 101; F4: 66). The cohort was divided into a discovery group (n = 151) and a validation group (n = 298). Logistic regression was performed to establish complex (FIBC3) and simplified (ABC3D) diagnostic scores that accurately identify advanced fibrosis. Performance for each was compared to established non-invasive fibrosis scoring systems. RESULTS: Plasma PRO-C3 levels correlated with grade of histological steatohepatitis (rs = 0.367, p ≪0.0001) and stage of fibrosis (rs = 0.462, p ≪0.0001), exhibiting similar performance to current fibrosis scores such as FIB4 for the detection of F≥3 fibrosis. FIBC3 exhibited substantially improved accuracy (AUROC 0.89 and 0.83 in the discovery and validation sets, respectively) and outperformed FIB4 and other similar diagnostic panels. The simplified version, ABC3D, was concurrently developed and had comparable diagnostic accuracy (AUROC 0.88 and 0.81 in the discovery and validation sets, respectively). CONCLUSION: Plasma PRO-C3 levels correlate with severity of steatohepatitis and fibrosis stage. The FIBC3 panel is an accurate tool with a single threshold value that maintains both sensitivity and specificity for the identification of F≥3 fibrosis in NAFLD, eliminating indeterminate results and outperforming commonly used non-invasive tools. A greatly simplified version (ABC3D) that is readily amenable to use in the clinic has been validated and shown to perform with similar accuracy, and may prove a useful tool in routine clinical practice. LAY SUMMARY: We performed a comprehensive, independent evaluation of a collagen biomarker (PRO-C3) to detect and quantify liver fibrosis in patients with non-alcoholic fatty liver disease (NAFLD). We report the development of 2 diagnostic panels using PRO-C3 to identify patients with advanced fibrosis, one optimal but more complex to calculate (FIBC3), the other easier to use (ABC3D) whilst still performing well.

Health-related Quality of Life in Nonalcoholic Fatty Liver Disease Associates With Hepatic Inflammation.

BACKGROUND & AIMS: Chronic liver disease has negative effects on health-related quality of life (HRQL). We analyzed data from the European non-alcoholic fatty liver disease (NAFLD) registry to assess the effects of NAFLD on HRQL. METHODS: We collected data from 304 patients (mean age, 52.3 ± 12.9 years) with histologically defined NAFLD enrolled prospectively into the European NAFLD Registry in Germany, the United Kingdom, and Spain. The chronic liver disease questionnaire (CLDQ) was completed within 6 months of liver biopsy collection. RESULTS: The mean CLDQ overall score was 5.0 ± 1.2, with the lowest score in the category fatigue (4.3 ± 1.6) and the highest scores for activity (5.4 ± 1.4). Women had significantly lower CLDQ scores than men (4.6 ± 1.3 vs 5.3 ± 1.1; P < .001). We found negative correlations between CLDQ scores and presence of obesity (P < .001), type 2 diabetes (P < .001), and dyslipidaemia (P < .01). There was a negative correlation between level of aspartate aminotransferase, but not alanine aminotransferase, and HRQL. Higher histological score of steatosis (1 vs 3) resulted in lower mean CLDQ score (5.3 ± 1.1 vs 4.5 ± 1.4; P < .01); higher level of lobular inflammation (0 vs 3) also resulted in lower mean CLDQ score (5.3 ± 1.2 vs 3.9 ± 1.8; P <. 001). In contrast, advanced fibrosis (F3-4) compared to early or intermediate fibrosis (F0-2) had no significant effect on mean CLDQ score (4.9 ± 1.2 vs 5.1 ± 1.3; P = .072). In multivariate analysis, patients sex, age, presence of type 2 diabetes, and inflammation were independently associated with low HRQL. CONCLUSION: In an analysis of data from the European NAFLD registry, we observed a substantial burden of symptoms in patients. In addition to age, sex, and the presence of diabetes, detection of lobular inflammation in biopsies correlated with lower HRQL.

The bidirectional impacts of alcohol consumption and the metabolic syndrome: Cofactors for progressive fatty liver disease.

Current medical practice artificially dichotomises a diagnosis of fatty liver disease into one of two common forms: alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD). Together, these account for the majority of chronic liver diseases worldwide. In recent years, there has been a dramatic increase in the prevalence of obesity and metabolic syndrome within the general population. These factors now coexist with alcohol consumption in a substantial proportion of the population. Each exposure sensitises the liver to the injurious effects of the other; an interaction that drives and potentially accelerates the genesis of liver disease. We review the epidemiological evidence and scientific literature that considers how alcohol consumption interacts with components of the metabolic syndrome to exert synergistic or supra-additive effects on the development and progression of liver disease, before discussing how these interactions may be addressed in clinical practice.

C-reactive protein as a predictor of low trough infliximab concentrations in patients who lose response to infliximab.

OBJECTIVE: Low serum infliximab concentrations are associated with an increased risk of loss of response in inflammatory bowel disease (IBD). The objective of this study was to evaluate the test characteristics of C-reactive protein (CRP) in identifying low serum infliximab concentrations in patients with IBD. METHODS: We measured serum infliximab concentrations and CRP levels in patients who experienced deteriorating symptoms while on infliximab (the reactive cohort). Receiver operating characteristic (ROC) curves were used to determine the CRP concentration threshold that identified an infliximab concentration <3 µg/mL at the time of loss of response. These CRP thresholds for infliximab concentration <3 µg/mL were then tested in a separate validation cohort. RESULTS: The reactive cohort contained 111 patients and the validation cohort contained 139 patients. In 41% of participants, serum infliximab concentration was <3 µg/mL. In the reactive cohort, the area under the ROC curve for CRP to identify an infliximab concentration <3 µg/mL was 0.70 (95% confidence interval [CI] 0.50-0.80, P = 0.02). A CRP level above 12 mg/L in the preceding 90 days provided a 90% specificity for the later detection of infliximab concentration <3 µg/mL. These test characteristics were similar in the validation cohort. CONCLUSION: CRP levels over 12 mg/L exhibit a high specificity for identifying patients with an infliximab concentration <3 µg/mL. CRP may be cost-effective for identifying patients with low concentrations of infliximab at the time of, or at risk of, loss of response.

WITHDRAWN: Epigenetics in Chronic Liver Disease.

This article has been withdrawn at the request of the editors. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.

High-risk family colorectal cancer screening service in Ireland: Critical review of clinical outcomes.

BACKGROUND: We present the 15-year experience of a family colorectal cancer screening service in Ireland with emphasis on real life experience and outcomes. METHODS: Questionnaires were used to assess family cancer history and assign patients to risk categories; 'Moderate Risk', HNPCC, (suspected) genetic syndrome (non-HNPCC), 'Low Risk'. Screening was by full colonoscopy. We report neoplastic yield, examining effect of risk category, age, gender, and index colonoscopy findings. RESULTS: Between 1998 and 2013, 2242 individuals were referred; 57.3% female, 42.7% male, median age 46 years (range9-85yrs). Median follow up time was 7.9yrs (range 0.5-15.3yrs). Follow up data after exclusion (non-compliance, known CRC) was available in 1496 (66.7%): 'Moderate risk' 785 (52.5%), HNPCC 256 (17.1%), (suspected) genetic syndrome (non-HNPCC) 85 (5.7%), 'Low Risk' 370 (24.7%). Screening was performed in 1025(68.5%) patients; colonoscopy data available for 993 (96.9%); total 1914 colonoscopies. At index colonoscopy, 178 (18.0%) patients had adenomas; 56 (5.5%) advanced adenoma. During the entire study period, 240 (24.2%) had an adenoma; 69 (7.0%) advanced adenoma. Cancers were diagnosed on screening in 2 patients. Older age and male gender were associated with higher adenoma detection rate; p<0.001, p=0.01, respectively. Risk category did not affect adenoma yield. Adenoma and advanced adenoma detection at index colonoscopy were associated with detection of same at follow up screening; p<0.001. CONCLUSION: Male gender and age (>50) were the core identifiable risk factors for neoplasia at screening colonoscopy in this family screening setting. Our results would support less intensive surveillance in younger patients (<50), particularly where index colonoscopy is normal.

Histology Grade Is Independently Associated With Relapse Risk in Patients With Ulcerative Colitis in Clinical Remission: A Prospective Study.

OBJECTIVES: Objective evidence of inflammation has been associated with the risk of relapse in patients with ulcerative colitis (UC) who are in clinical remission. We compared endoscopic and histologic grades for their ability to predict clinical relapse in this patient population. METHODS: Patients with UC in clinical remission were prospectively enrolled into an observational cohort. Baseline endoscopic scores (Mayo) and histological (Geboes) grades and blood markers were collected. All subjects were followed for 12 months and relapse determined using clinical indices. RESULTS: A total of 179 subjects were enrolled into the study and followed for 12 months. Clinical relapse occurred in 23%; 5% were hospitalized, and 2% underwent colectomy. In univariate analysis, the baseline Mayo endoscopy score and the Geboes histology grade were significantly associated with the later development of clinical relapse (P<0.001 for both), but only the histology grade remained significant in a multivariate model (P=0.006). The relative risk of clinical relapse was 3.5 (95% CI 1.9-6.4, P<0.0001) in subjects whose baseline Geboes grade was ≥3.1. The area under the curve was 0.73 for the Geboes histology grade to identify subjects at risk of future clinical relapse. Of the patients in clinical, endoscopic, and histological remission at baseline (n=82), only 7% had a clinical relapse over the subsequent 12 months. CONCLUSIONS: Histology grade has the strongest association with the risk of clinical relapse in patients with UC who are in clinical remission. Consideration should be given to including this end point in evaluating therapy for UC.

Adherence to Rectal Mesalamine in Patients with Ulcerative Colitis.

BACKGROUND: Rectal mesalamine is an effective induction and maintenance therapy for ulcerative colitis. Little is known about the adherence rates to rectal mesalamine or barriers to its use. The aim was to quantify the prevalence of nonadherence to rectal mesalamine and to identify patient-reported barriers to adherence. METHODS: A cohort of patients with ulcerative colitis was prospectively enrolled in this observational study and followed for 12 months. Adherence was assessed by tracking pharmacy refills (medication possession ratio). Individual interviews were undertaken in a subset of subjects. Transcripts from the focus groups and interviews were analyzed to identify themes and links between these themes using qualitative data software (MaxQDA). RESULTS: Seventy patients prescribed rectal mesalamine were prospectively enrolled in the study. At enrollment, 39 of 70 subjects (55%) self-reported "occasional nonadherence" to rectal mesalamine. Over the 12-month follow-up period, only 20 subjects (26%) completed 3 or more refills. Males, or subjects prescribed a once-a-day suppository, were significantly more likely to refill than females (odds ratio = 3.3, 95% confidence interval, 1.1-10.9) or those prescribed suppositories more than once a day (odds ratio = 1.3, 95% confidence interval, 1.1-1.7). By medication possession ratio criteria, 71% of all subjects were nonadherent with their prescribed regimen (medication possession ratio <0.6). Nonadherers were significantly older than adherent subjects: mean age 48 years in nonadherers, versus 37 in adherers, P = 0.04. Patients who were nonadherent to rectal mesalamine frequently cited the mode of administration (65%) and busy lifestyle (40%) as reasons for nonadherence. CONCLUSIONS: Intentional nonadherence is common in patients who have been prescribed rectal mesalamine. Gender, age, frequency of dosing, and lifestyle factors may impact adherence.

Position of the academy of nutrition and dietetics: nutrition security in developing nations: sustainable food, water, and health.

It is the position of the Academy of Nutrition and Dietetics that all people should have consistent access to an appropriately nutritious diet of food and water, coupled with a sanitary environment, adequate health services, and care that ensure a healthy and active life for all household members. The Academy supports policies, systems, programs, and practices that work with developing nations to achieve nutrition security and self-sufficiency while being environmentally and economically sustainable. For nations to achieve nutrition security, all people must have access to a variety of nutritious foods and potable drinking water; knowledge, resources, and skills for healthy living; prevention, treatment, and care for diseases affecting nutrition status; and safety-net systems during crisis situations, such as natural disasters or deleterious social and political systems. More than 2 billion people are micronutrient deficient; 1.5 billion people are overweight or obese; 870 million people have inadequate food energy intake; and 783 million people lack potable drinking water. Adequate nutrient intake is a concern, independent of weight status. Although this article focuses on nutritional deficiencies in developing nations, global solutions for excesses and deficiencies need to be addressed. In an effort to achieve nutrition security, lifestyles, policies, and systems (eg, food, water, health, energy, education/knowledge, and economic) contributing to sustainable resource use, environmental management, health promotion, economic stability, and positive social environments are required. Food and nutrition practitioners can get involved in promoting and implementing effective and sustainable policies, systems, programs, and practices that support individual, community, and national efforts.

Influence of stromal-epithelial interactions on breast cancer in vitro and in vivo.

Stromal cell-secreted chemokines including CCL2 have been implicated in the primary tumor microenvironment, as mediators of tumor cell migration, proliferation, and angiogenesis. Expression of CCL2 and its principal receptor CCR2 was analyzed by RQ-PCR in primary tumor cells and breast cancer cell lines. Breast cancer cell lines (MDA-MB-231, T47D) were co-cultured directly on a monolayer of primary breast tumor and normal stromal cells, retrieved using EpCAM+ magnetic beads, and changes in expression of CCL2, CCR2, MMP11, ELK1, VIL2, and Ki67 detected by RQ-PCR. Epithelial cell migration and proliferation in response to stromal cell-secreted factors was also analyzed. In vivo, tumor xenografts were formed by co-injecting T47D cells with primary tumor stromal cells. Following establishment, tumors were harvested and digested, epithelial cells retrieved and analyzed by RQ-PCR. Whole tumor tissue was also analyzed by immunohistochemistry for CD31 and the VIL2 encoded protein Ezrin. Tumor stromal cells expressed significantly higher levels of CCL2 than normal cells, with no CCR2 expression detected. Primary epithelial cells and breast cancer cell lines expressed elevated CCL2, with relative expression of CCR2 found to be higher than the ligand. Interaction of breast cancer epithelial cells with primary tumor, but not normal stromal cells, stimulated increased expression of CCL2 (8-fold), ELK1 (6-fold), VIL2 (6-fold), and MMP11 (17-fold). Factors secreted by stromal cells, including CCL2, stimulated a significant increase in epithelial cell migration, with no effect on cell proliferation in vitro observed. In vivo, the presence of stromal cells resulted in tumors of increased volume, mediated at least in part through neoangiogenesis demonstrated by immunohistochemistry (CD31). Admixed tumor xenografts exhibited increased expression of Ki67, MMP11, VIL2, and ELK1. Elevated Ezrin protein was also detected, with increased cytoplasmic localization. The results presented highlight mechanisms through which breast cancer epithelial cells can harness stromal cell biology to support tumor progression.