Development of an affordable, immersive model for robotic vaginal cuff closure: a randomized trial.

Safe and secure closure of the vaginal cuff is a critical component of a robotic assisted hysterectomy procedure. Our aim in this study is to develop and validate a novel vaginal cuff closure model (VC) created from porcine heart that allows trainees to obtain competency in a low-risk environment. Ten expert and 20 novice robotic surgeons performed a cuff closure exercise on the VC model and on the dV-Trainer®, a virtual reality simulator (VR). Performances were timed, videotaped, and scored using the modified Global Evaluative Assessment of Robotic Skills (mGEARS) score. Expert robotic surgeons completed the task faster on both the VR (531 vs. 814 s, p = 0.03) and the VC platforms (311 vs. 631 s, p < 0.001) and achieved higher mGEAR scores (32.25 vs. 22.07, p < 0.0001). Knot quality and suturing accuracy were better in the VC than in the VR environment in both groups. In a post-completion survey, both expert and novice surgeons expressed strong preference towards the VC model. In this study, the novel VC model proved to be a reliable simulation tool with high face, content, and construct validity. Due to its simplicity and low cost, this high-yield simulation exercise can easily be incorporated into robotic training curricula of obstetrics and gynecology residents.

Whole Cell MALDI Fingerprinting Is a Robust Tool for Differential Profiling of Two-Component Mammalian Cell Mixtures.

MALDI fingerprinting was first described two decades ago as a technique to identify microbial cell lines. Microbial fingerprinting has since evolved into an automated platform for microorganism identification and classification, which is now routinely used in clinical and environmental sectors. The extension of fingerprinting to mammalian cells has yet to progress partly due to compartmentalization of eukaryotic cells and overall higher cellular complexity. A number of publications on mammalian whole cell fingerprinting suggest that the method could be useful for classification of different cell types, cell states, and monitoring cell differentiation. We report the optimization of MALDI fingerprinting workflow parameters for mammalian cells and its application for differential profiling of mammalian cell lines and two-component cell line mixtures. Murine fallopian tube cells and high-grade ovarian carcinoma cell lines and their mixtures are used as model mammalian cell lines. Two-component cell mixtures serve to determine the method's feasibility for complex biological samples as the ability to detect cancer cells in a mixed cell population. The level of detection of cancer cells in the two-component mixture by principle component analysis (PCA) starts to deteriorate at 5% but with application of a different statistical approach, Wilcoxon rank sum test, the level of detection was determined to be 1%. The ability to differentiate heterogeneous cell mixtures will help further extend whole cell MALDI fingerprinting to complex biological systems. Graphical Abstract.

Adipocyte-induced CD36 expression drives ovarian cancer progression and metastasis.

Ovarian cancer (OvCa) is characterized by widespread and rapid metastasis in the peritoneal cavity. Visceral adipocytes promote this process by providing fatty acids (FAs) for tumour growth. However, the exact mechanism of FA transfer from adipocytes to cancer cells remains unknown. This study shows that OvCa cells co-cultured with primary human omental adipocytes express high levels of the FA receptor, CD36, in the plasma membrane, thereby facilitating exogenous FA uptake. Depriving OvCa cells of adipocyte-derived FAs using CD36 inhibitors and short hairpin RNA knockdown prevented development of the adipocyte-induced malignant phenotype. Specifically, inhibition of CD36 attenuated adipocyte-induced cholesterol and lipid droplet accumulation and reduced intracellular reactive oxygen species (ROS) content. Metabolic analysis suggested that CD36 plays an essential role in the bioenergetic adaptation of OvCa cells in the adipocyte-rich microenvironment and governs their metabolic plasticity. Furthermore, the absence of CD36 affected cellular processes that play a causal role in peritoneal dissemination, including adhesion, invasion, migration and anchorage independent growth. Intraperitoneal injection of CD36-deficient cells or treatment with an anti-CD36 monoclonal antibody reduced tumour burden in mouse xenografts. Moreover, a matched cohort of primary and metastatic human ovarian tumours showed upregulation of CD36 in the metastatic tissues, a finding confirmed in three public gene expression data sets. These results suggest that omental adipocytes reprogram tumour metabolism through the upregulation of CD36 in OvCa cells. Targeting the stromal-tumour metabolic interface via CD36 inhibition may prove to be an effective treatment strategy against OvCa metastasis.

Mesothelial cells promote early ovarian cancer metastasis through fibronectin secretion.

Ovarian cancer (OvCa) metastasizes to organs in the abdominal cavity, such as the omentum, which are covered by a single layer of mesothelial cells. Mesothelial cells are generally thought to be "bystanders" to the metastatic process and simply displaced by OvCa cells to access the submesothelial extracellular matrix. Here, using organotypic 3D cultures, we found that primary human mesothelial cells secrete fibronectin in the presence of OvCa cells. Moreover, we evaluated the tumor stroma of 108 human omental metastases and determined that fibronectin was consistently overexpressed in these patients. Blocking fibronectin production in primary mesothelial cells in vitro or in murine models, either genetically (fibronectin 1 floxed mouse model) or via siRNA, decreased adhesion, invasion, proliferation, and metastasis of OvCa cells. Using a coculture model, we determined that OvCa cells secrete TGF-ß1, which in turn activates a TGF-ß receptor/RAC1/SMAD-dependent signaling pathway in the mesothelial cells that promotes a mesenchymal phenotype and transcriptional upregulation of fibronectin. Additionally, blocking α5 or ß1 integrin function with antibodies reduced metastasis in an orthotopic preclinical model of OvCa metastasis. These findings indicate that cancer-associated mesothelial cells promote colonization during the initial steps of OvCa metastasis and suggest that mesothelial cells actively contribute to metastasis.

Expression of the homeobox gene HOXA9 in ovarian cancer induces peritoneal macrophages to acquire an M2 tumor-promoting phenotype.

Tumor-associated macrophages (TAMs) exhibit an M2 macrophage phenotype that suppresses anti-tumor immune responses and often correlates with poor outcomes in patients with cancer. Patients with ovarian cancer frequently present with peritoneal carcinomatosis, but the mechanisms that induce naïve peritoneal macrophages into TAMs are poorly understood. In this study, we found an increased abundance of TAMs in mouse i.p. xenograft models of ovarian cancer that expressed HOXA9, a homeobox gene that is associated with poor prognosis in patients with ovarian cancer. HOXA9 expression in ovarian cancer cells stimulated chemotaxis of peritoneal macrophages and induced macrophages to acquire TAM-like features. These features included induction of the M2 markers, CD163 and CD206, and the immunosuppressive cytokines, IL-10 and chemokine ligand 17, and down-regulation of the immunostimulatory cytokine, IL-12. HOXA9-mediated induction of TAMs was primarily due to the combinatorial effects of HOXA9-induced, tumor-derived transforming growth factor-ß2 and chemokine ligand 2 levels. High HOXA9 expression in clinical specimens of ovarian cancer was strongly associated with increased abundance of TAMs and intratumoral T-regulatory cells and decreased abundance of CD8(+) tumor-infiltrating lymphocytes. Levels of immunosuppressive cytokines were also elevated in ascites fluid of patients with tumors that highly expressed HOXA9. HOXA9 may, therefore, stimulate ovarian cancer progression by promoting an immunosuppressive microenvironment via paracrine effects on peritoneal macrophages.

HOXA9 promotes ovarian cancer growth by stimulating cancer-associated fibroblasts.

Epithelial ovarian cancers (EOCs) often exhibit morphologic features of embryonic Müllerian duct-derived tissue lineages and colonize peritoneal surfaces that overlie connective and adipose tissues. However, the mechanisms that enable EOC cells to readily adapt to the peritoneal environment are poorly understood. In this study, we show that expression of HOXA9, a Müllerian-patterning gene, is strongly associated with poor outcomes in patients with EOC and in mouse xenograft models of EOC. Whereas HOXA9 promoted EOC growth in vivo, HOXA9 did not stimulate autonomous tumor cell growth in vitro. On the other hand, expression of HOXA9 in EOC cells induced normal peritoneal fibroblasts to express markers of cancer-associated fibroblasts (CAFs) and to stimulate growth of EOC and endothelial cells. Similarly, expression of HOXA9 in EOC cells induced normal adipose- and bone marrow-derived mesenchymal stem cells (MSCs) to acquire features of CAFs. These effects of HOXA9 were due in substantial part to its transcriptional activation of the gene encoding TGF-ß2 that acted in a paracrine manner on peritoneal fibroblasts and MSCs to induce CXCL12, IL-6, and VEGF-A expression. These results indicate that HOXA9 expression in EOC cells promotes a microenvironment that is permissive for tumor growth.

Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth.

Intra-abdominal tumors, such as ovarian cancer, have a clear predilection for metastasis to the omentum, an organ primarily composed of adipocytes. Currently, it is unclear why tumor cells preferentially home to and proliferate in the omentum, yet omental metastases typically represent the largest tumor in the abdominal cavities of women with ovarian cancer. We show here that primary human omental adipocytes promote homing, migration and invasion of ovarian cancer cells, and that adipokines including interleukin-8 (IL-8) mediate these activities. Adipocyte-ovarian cancer cell coculture led to the direct transfer of lipids from adipocytes to ovarian cancer cells and promoted in vitro and in vivo tumor growth. Furthermore, coculture induced lipolysis in adipocytes and ß-oxidation in cancer cells, suggesting adipocytes act as an energy source for the cancer cells. A protein array identified upregulation of fatty acid-binding protein 4 (FABP4, also known as aP2) in omental metastases as compared to primary ovarian tumors, and FABP4 expression was detected in ovarian cancer cells at the adipocyte-tumor cell interface. FABP4 deficiency substantially impaired metastatic tumor growth in mice, indicating that FABP4 has a key role in ovarian cancer metastasis. These data indicate adipocytes provide fatty acids for rapid tumor growth, identifying lipid metabolism and transport as new targets for the treatment of cancers where adipocytes are a major component of the microenvironment.

Targeting the urokinase plasminogen activator receptor inhibits ovarian cancer metastasis.

PURPOSE: To understand the functional and preclinical efficacy of targeting the urokinase plasminogen activator receptor (u-PAR) in ovarian cancer. EXPERIMENTAL DESIGN: Expression of u-PAR was studied in 162 epithelial ovarian cancers, including 77 pairs of corresponding primary and metastatic tumors. The effect of an antibody against u-PAR (ATN-658) on proliferation, adhesion, invasion, apoptosis, and migration was assessed in 3 (SKOV3ip1, HeyA8, and CaOV3) ovarian cancer cell lines. The impact of the u-PAR antibody on tumor weight, number, and survival was examined in corresponding ovarian cancer xenograft models and the mechanism by which ATN-658 blocks metastasis was explored. RESULTS: Only 8% of all ovarian tumors were negative for u-PAR expression. Treatment of SKOV3ip1, HeyA8, and CaOV3 ovarian cancer cell lines with the u-PAR antibody inhibited cell invasion, migration, and adhesion. In vivo, anti-u-PAR treatment reduced the number of tumors and tumor weight in CaOV3 and SKOV3ip1 xenografts and reduced tumor weight and increased survival in HeyA8 xenografts. Immunostaining of CaOV3 xenograft tumors and ovarian cancer cell lines showed an increase in active-caspase 3 and TUNEL staining. Treatment with u-PAR antibody inhibited α(5)-integrin and u-PAR colocalization on primary human omental extracellular matrix. Anti-u-PAR treatment also decreased the expression of urokinase, u-PAR, ß(3)-integrin, and fibroblast growth factor receptor-1 both in vitro and in vivo. CONCLUSIONS: This study shows that an antibody against u-PAR reduces metastasis, induces apoptosis, and reduces the interaction between u-PAR and α(5)-integrin. This provides a rationale for targeting the u-PAR pathway in patients with ovarian cancer and for further testing of ATN-658 in this indication.

Subcutaneous methylnaltrexone to restore postoperative bowel function in a long-term opiate user.

INTRODUCTION: One of the most common undesired effects of analgesic opioid use and addiction is constipation. Numerous pharmacologic agents have been used to treat opioid-induced bowel hypomotility with limited success. Methylnaltrexone bromide (MNTX) selectively targets the peripheral adverse effects of opioids while preserving the central analgesic effects of opioid agonist treatment. CASE: While it is indicated for use in nonsurgical patients in the palliative care setting, here we report the use of MNTX for the alleviation of postoperative ileus in a heroin user with recurrent cervical cancer undergoing diverting colostomy and urinary conduit placement. CONCLUSIONS: Results suggest that MNTX may accelerate postoperative gastrointestinal recovery in opioid-dependent patients. Further studies are warranted to evaluate its role in the pharmacologic management of postoperative ileus.

Case study of the morphologic variation of circulating tumor cells.

We report a detailed cytomorphologic evaluation of the circulating component of widely metastatic breast carcinoma. A previously healthy 38-year-old woman was diagnosed with breast cancer. Wide local excision revealed a 1.7-cm infiltrating ductal adenocarcinoma, BSR score 7/9 with angiolymphatic invasion, and 4/20 lymph nodes positive for carcinoma. Five years later, a bone marrow biopsy revealed involvement of bone marrow by metastatic breast carcinoma, and shortly thereafter, metastases were identified in the liver and lung hilum. She enrolled in a clinical investigation for the detection of circulating tumor cells (CTCs) in breast carcinoma. A total of 659 CTCs were identified in a 10-mL blood sample using an immunofluorescent protocol targeting cytokeratins and detected using fiber-optic array scanning technology. The detected CTCs were subsequently stained with a Wright-Giemsa stain, and representative cells were evaluated in detail by light microscopy for morphologic evaluation. We find that the patient's CTCs exhibit a high degree of pleomorphism including CTCs with high and low nuclear-to-cytoplasmic ratios along with CTCs exhibiting early and late apoptotic changes. In addition, in comparison with her tumor cells in other sites, the full morphologic spectrum of cancer cells present in primary and metastatic tumor is also present in peripheral blood circulation.

Laser microdissection in translational and clinical research.

Laser microdissection (LMD) is now a well established method for isolating individual cells or subcellular structures from a heterogeneous cell population. In recent years, cell, DNA, RNA, and protein based techniques has been successfully coupled to LMD and important information has been gathered through the analysis of the genome, transcriptome, and more recently the proteome of individual microdissected cells. The aims of this review are to summarize and compare the principles of different laser microdissection instruments and techniques, to discuss sample preparation procedures for microdissection, and to provide wide variety of examples of translational/clinical research applications of LMD. Novel techniques specifically developed for the improved isolation of stained cells, living cells, or rare cells are also discussed.LMD has become an indispensable tool in the preparation of homogenous samples for sophisticated cell or molecular assays. Despite major technological advances, the labor requirements of LMD are still relatively high. However, understanding the advantages and disadvantages of LMD technology and associated sample preparation procedures may aid in the earlier introduction of this method into the routine clinical diagnostics.

High speed detection of circulating tumor cells.

Epithelial tumor cells circulate in peripheral blood at ultra-low concentrations in cancer patients. We have developed an instrument capable of rapid and accurate detection of rare cells in circulation utilizing fiber-optic array scanning technology (FAST). The FAST cytometer can locate immunofluorescently labeled rare cells on glass substrates at scan rates 500 times faster than conventional automated digital microscopy. These high scan rates are achieved by collecting fluorescent emissions using a fiber bundle with a large (50 mm) field of view. Very high scan rates make possible the ability to detect rare events without the requirement for an enrichment step. The FAST cytometer was used to detect, image and re-image circulating tumor cells in peripheral blood of breast cancer patients. This technology has the potential to serve as a clinically useful point-of-care diagnostic and a prognostic tool for cancer clinicians. The use of a fixed substrate permits the re-identification and re-staining of cells allowing for additional morphologic and biologic information to be obtained from previously collected and identified cells.

A rare-cell detector for cancer.

Although a reliable method for detection of cancer cells in blood would be an important tool for diagnosis and monitoring of solid tumors in early stages, current technologies cannot reliably detect the extremely low concentrations of these rare cells. The preferred method of detection, automated digital microscopy (ADM), is too slow to scan the large substrate areas. Here we report an approach that uses fiber-optic array scanning technology (FAST), which applies laser-printing techniques to the rare-cell detection problem. With FAST cytometry, laser-printing optics are used to excite 300,000 cells per sec, and emission is collected in an extremely wide field of view, enabling a 500-fold speed-up over ADM with comparable sensitivity and superior specificity. The combination of FAST enrichment and ADM imaging has the performance required for reliable detection of early-stage cancer in blood.

Reliable and sensitive identification of occult tumor cells using the improved rare event imaging system.

PURPOSE: The purpose of this study was to assess the feasibility of using rare event imaging system (REIS)-assisted analysis to detect occult tumor cells (OTCs) in peripheral blood (PB). The study also sought to determine whether REIS-assisted OTC detection presents a clinically viable alternative to manual microscopic detection to establish the true significance of OTC from solid epithelial tumors. EXPERIMENTAL DESIGN: We recently demonstrated proof of concept using a fluorescence-based automated microscope system, REIS, for OTC detection from the PB. For this study, the prototype of the system was adopted for high-throughput and high-content cellular analysis. RESULTS: The performance of the improved REIS was examined using normal blood (n = 10), normal blood added to cancer cells (n = 20), and blood samples obtained from cancer patients (n = 80). Data from the screening of 80 clinical slides from breast and lung cancer patients, by manual microscopy and by the REIS, revealed that as many as 14 of 35 positive slides (40%) were missed by manual screening but positively identified by REIS. In addition, REIS-assisted scanning reliably and reproducibly quantified the total number of cells analyzed in the assay and categorized positive cells based on their marker expression profile. CONCLUSIONS: REIS-assisted analysis provides excellent sensitivity and reproducibility for OTC detection. This approach may enable an improved method for screening of PB samples and for obtaining novel information about disease staging and about risk evaluation in cancer patients.

Automated detection of immunofluorescently labeled cytomegalovirus-infected cells in isolated peripheral blood leukocytes using decision tree analysis.

BACKGROUND: Cytomegalovirus (CMV) infection continues to be a major problem for immunocompromised patients. Detection of viral antigens in leukocytes (antigenemia assay) is widely used for the diagnosis of CMV infection and for guiding antiviral therapy. The antigenemia technique, contingent upon the manual microscopic analysis of rare cells, is a laborious task that is subject to human error. In this study, we combine automated microscopy with artificial intelligence for reliable detection of fluorescently labeled CMV-infected cells. METHODS: Cytospin preparations of peripheral blood leukocytes were immunofluorescently labeled for the CMV lower matrix phosphoprotein (pp65) and scanned in the Rare Event Imaging System (REIS), a fully automated image cytometer. The REIS detected potential positive objects and digitally recorded 49 measured cellular features for each identified case. The measurement data of these objects were analyzed by the See5 decision tree (DT) algorithm to ascertain whether they were true-positive detections. RESULTS: The DT was built from the measurement data of 2,047 true- and 2,028 false-positive detections, collected from 32 patient samples. By designating misclassifications of false-negatives three times more costly, the 10-fold cross-validation sensitivity, specificity, and misclassification error of the assay was 94.3%, 56.2%, and 25%, respectively. The method was also validated using an independent test set of 21 patient samples, in which similar results were obtained. CONCLUSIONS: To our knowledge, this study represents the first attempt to improve the accuracy of rare event image cytometry through the implementation of artificial intelligence methodology. Results suggest that cost-sensitive decision tree analysis of digitally measured cellular features vastly improves the performance of rare event image cytometry.

Circulating tumor cells and serum tumor biomarkers in small cell lung cancer.

Lung cancer accounts for approximately 30% of all cancer mortalities in the United States. Small cell lung cancer (SCLC), which is an aggressive malignancy with frequent and early metastases, accounts for about 15% of all of the lung cancer cases with a dismal 5-year survival rate of < 5% with current standard therapies. Early detection of SCLC is challenging, in part due to the lack of adequate serum tumor markers. The goal of this review is to summarize the current knowledge of circulating tumor cells and serum biomarkers in small cell lung cancer. The role of circulating tumor cells in prognostication is controversial, but may be better defined with advancing technologies of detection of such cells with higher precision, and improved clinico-pathological correlations. The current knowledge on the known serum cytokines and tumor biomarkers of SCLC, such as CEA, chromogranin-A and neuron-specific enolase will be presented. Serum cytokines, such as vascular endothelial growth factor (VEGF), stem cell factor (SCF) and hepatocyte growth factor/scatter factor (HGF/SF) are also discussed. New findings in the search for novel diagnostic and therapeutic molecular markers using the emerging genomics and proteomics technologies are emphasized. It is our hope that validation of these new research platforms and technologies will result in improved early detection, prognostication and finally treatment of SCLC with potential novel molecularly-targeted therapeutics.