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Supplemental Methods, Supplemental Tables 1, 3, 4, 6, 7, 8, Supplemental Figures 1-12 Supplemental Table 1. Overall patient characteristics. Supplemental Table 3. Patient Characteristics by lymph node molecular classification. Supplemental Table 4. Univariate and multivariate analysis for locoregional control. Supplemental Table 7. Accuracy of existing classifiers to predict Group 2 LNM subtype. Supplemental Table 8. 73 gene classifier Supplemental Figure 1. Patient selection. Supplemental Figure 2. Metrics of the 73 gene classifier. (A) Metrics of 73 gene classifier. (B) Misclassification error for C2 and C13 groups. (C) Misclassification of entire classifier. Supplemental Figure 3. Outcomes in HNSCC cohort.Kaplan-Meier analysis of: (A) Locoregional control. (B) Local-only control. (C) Relapse free survival. (D) Regional control. (E) Distant control. (F) Overall survival. Supplemental Figure 4. Outcomes by molecular subtypes of LNMs.Kaplan-Meier analysis of: (A) Regional control. (B) Distant control. Supplementary Figure 5: DEmiRNAsin LNMs subtypes. Supplemental Figure 6. Consensus clustering of pairwise DEGs for other predefined cluster numbers does not identify other LNMs subtypes that predict for disease recurrence. Supplemental Figure 7. Enrichment of KEGG pathways in LNMs subtypes. Supplemental Figure 8. Estimated abundance of immune cells. Supplemental Figure 9. LNMs subtypes cluster separately from non-metastatic lymph nodes in the same cohort. Supplemental Figure 10. LNMs subtypes cluster separately from normal lymph nodes derived from publically available microarray databases. Supplemental Figure 11. The transcriptome of lymph node metastasis better predicts for patients at higher risk of relapse or death. Supplemental Figure 12. Receiver-Operator Curve for 73 gene LNM-classifier.

Gene Methylation Detection, Sensitivity, Specificity Using Detectable vs. Non-detectable cutoff in blacks

Supplemental Table 2. LN classification by iCluster and PAM classifier.

Supplemental Table 5. DEmRNAs and DEmiRNAs in LNMs.

Purpose:Low-dose CT screening can reduce lung cancer–related mortality. However, CT screening has an FDR of nearly 96%. We sought to assess whether urine samples can be a source for DNA methylation–based detection of non–small cell lung cancer (NSCLC).Experimental Design:This nested case–control study of subjects with suspicious nodules on CT imaging obtained plasma and urine samples preoperatively. Cases (n = 74) had pathologic confirmation of NSCLC. Controls (n = 27) had a noncancer diagnosis. We detected promoter methylation in plasma and urine samples using methylation on beads and quantitative methylation–specific real-time PCR for cancer-specific genes (CDO1, TAC1, HOXA7, HOXA9, SOX17, and ZFP42).Results:DNA methylation at cancer-specific loci was detected in both plasma and urine, and was more frequent in patients with cancer compared with controls for all six genes in plasma and in CDO1, TAC1, HOXA9, and SOX17 in urine. Univariate and multivariate logistic regression analysis showed that methylation detection in each one of six genes in plasma and CDO1, TAC1, HOXA9, and SOX17 in urine were significantly associated with the diagnosis of NSCLC, independent of age, race, and smoking pack-years. When methylation was detected for three or more genes in both plasma and urine, the sensitivity and specificity for lung cancer diagnosis were 73% and 92%, respectively.Conclusions:DNA methylation–based biomarkers in plasma and urine could be useful as an adjunct to CT screening to guide decision-making regarding further invasive procedures in patients with pulmonary nodules.

Supplemental Table 6: mRNA:miRNA pairs enriched in LNMs subtypes.

Purpose:In advanced stage head and neck squamous cell cancers (HNSCC), approximately half of the patients with lymph node metastases (LNM) are not cured. Given the heterogeneous outcomes in these patients, we profiled the expression patterns of LNMs to identify the biological factors associated with patient outcomes.Experimental Design: We performed mRNAseq and miRNAseq on 72 LNMs and 29 matched primary tumors from 34 patients with HNSCC. Clustering identified molecular subtypes in LNMs and in primary tumors. Prediction Analysis of Microarrays algorithm identified a 73-gene classifier that distinguished LNM subtypes. Gene-set enrichment analysis identified pathways upregulated in LNM subtypes.Results:Integrative clustering identified three distinct LNM subtypes: (i) an immune subtype (Group 1), (ii) an invasive subtype (Group 2), and (iii) a metabolic/proliferative subtype (Group 3). Group 2 subtype was associated with significantly worse locoregional control and survival. LNM-specific subtypes were not observed in matched primary tumor specimens. In HNSCCs, breast cancers, and melanomas, a 73-gene classifier identified similar Group 2 LNM subtypes that were associated with worse disease control and survival only when applied to lymph node sites, but not when applied to other primary tumors or metastatic sites. Similarly, previously proposed prognostic classifiers better distinguished patients with worse outcomes when applied to the transcriptional profiles of LNMs, but not the profiles of primary tumors.Conclusions:The transcriptional profiles of LNMs better predict outcomes than transcriptional profiles of primary tumors. The LNMs display site-specific subtypes associated with worse disease control and survival across multiple cancer types.

Supplemental Table 9. Breast cancer characteristics

Supplemental Table 10. Melanoma characteristics.

Tyrosine kinase inhibitors were found to be clinically effective for treatment of patients with certain subsets of cancers carrying somatic mutations in receptor tyrosine kinases. However, the duration of clinical response is often limited, and patients ultimately develop drug resistance. Here, we use single-cell RNA sequencing to demonstrate the existence of multiple cancer cell subpopulations within cell lines, xenograft tumors and patient tumors. These subpopulations exhibit epigenetic changes and differential therapeutic sensitivity. Recurrently overrepresented ontologies in genes that are differentially expressed between drug tolerant cell populations and drug sensitive cells include epithelial-to-mesenchymal transition, epithelium development, vesicle mediated transport, drug metabolism and cholesterol homeostasis. We show analysis of identified markers using the LINCS database to predict and functionally validate small molecules that target selected drug tolerant cell populations. In combination with EGFR inhibitors, crizotinib inhibits the emergence of a defined subset of EGFR inhibitor-tolerant clones. In this study, we describe the spectrum of changes associated with drug tolerance and inhibition of specific tolerant cell subpopulations with combination agents., It has been proposed that resistance to targeted therapies in non-small cell lung carcinoma (NSCLC) is due to a nonhomogeneous cell population. Here the authors analyse preclinical NSCLC models using single-cell RNA-seq and identify drug tolerant cell states and subpopulations, as well as associated genes.

Background: Technologies related to the establishment of primary tumor cell cultures from solid tumors, including glioblastoma, are increasingly important to oncology research and practice. However, processing of fresh tumor specimens for establishment of primary cultures on the day of surgical collection is logistically difficult. The feasibility of viable cryopreservation of glioblastoma specimens, allowing for primary culture establishment weeks to months after surgical tumor collection and freezing, was demonstrated by Mullins et al. in 2013, with a success rate of 59% that was not significantly lower than that achieved with fresh tumor tissue. However, research targeting optimization of viable glioblastoma cryopreservation protocols for establishment of primary tumor cultures has been limited. Objectives: The objective of this study was to optimize glioblastoma cryopreservation methods for viable cryobanking and to determine if two-dimensional (2D) or three-dimensional (3D) culture conditions were more supportive of glioblastoma growth after thawing of frozen tumor specimens. Methods: Portions of eight human glioblastoma specimens were cryopreserved by four different protocols differing in the time of enzymatic digestion (before or after cryopreservation), and in the type of cryopreservation media (CryoStor CS10 or 10% dimethyl sulfoxide and 90% fetal calf serum). After 1 month, frozen tissues were thawed, enzymatically digested, if not digested before, and used for initiation of 2D or 3D primary tumor cultures to determine viability. Results: Among the tested cryopreservation and culturing protocols, the most efficient combinations of cryopreservation and culture were those associated with the use of CryoStor CS10 cryopreservation medium, enzymatic digestion before freezing, and 2D culturing after thawing with a successful culture rate of 8 out of 8 cases (100%). Two-dimensional cultures were in general more efficient for the support of tumor cell growth after thawing than 3D cultures. Conclusions: This study supports development of evidence-based viable glioblastoma cryopreservation methods for use in glioblastoma biobanking and research.

Results from epidemiological and prospective studies indicate a close association between periodontitis and diabetes. However the mechanisms by which periodontal pathogens influence the development of prediabetes/diabetes are not clear. We previously reported that oral administration of a periodontal pathogen, Porphyromonas gingivalis (Pg) to WT mice results in insulin resistance, hyperinsulinemia, and glucose intolerance and that Pg translocates to the pancreas. In the current study, we determined the specific localization of Pg in relation to mouse and human pancreatic α- and β-cells using 3-D confocal and immunofluorescence microscopy and orthogonal analyses. Pg/gingipain is intra- or peri-nuclearly localized primarily in β-cells in experimental mice and also in human post-mortem pancreatic samples. We also identified bihormonal cells in experimental mice as well as human pancreatic samples. A low percentage of bihormonal cells has intracellular Pg in both humans and experimental mice. Our data show that the number of Pg translocated to the pancreas correlates with the number of bihormonal cells in both mice and humans. Our findings suggest that Pg/gingipain translocates to pancreas, particularly β-cells in both humans and mice, and this is strongly associated with emergence of bihormonal cells.

Background Anal squamous cell carcinoma (SCC) generally carries a favorable prognosis, as most tumors are highly sensitive to standard of care chemoradiation. However, outcomes are poor for the 20–30% of patients who are refractory to this approach, and many will require additional invasive procedures with no guarantee of disease resolution. Methods To identify the patients who are unlikely to respond to the current standard of care chemoradiation protocol, we explored a variety of objective clinical findings as a potential predictor of treatment failure and/or mortality in a single center retrospective study of 42 patients with anal SCC. Results Patients with an increase in total peripheral white blood cells (WBC) and/or neutrophils (ANC) had comparatively poor clinical outcomes, with increased rates of death and treatment failure, respectively. Using pre-treatment biopsies from 27 patients, tumors with an inflamed, neutrophil dominant stroma also had poor therapeutic responses, as well as reduced overall and disease-specific survival. Following chemoradiation, we observed uniform reductions in nearly all peripheral blood leukocyte subtypes, and no association between peripheral white blood cells and/or neutrophils and clinical outcomes. Additionally, post-treatment biopsies were available from 13 patients. In post-treatment specimens, patients with an inflamed tumor stroma now demonstrated improved overall and disease-specific survival, particularly those with robust T-cell infiltration. Conclusions Combined, these results suggest that routinely performed leukocyte subtyping may have utility in risk stratifying patients for treatment failure in anal SCC. Specifically, pre-treatment patients with a high WBC, ANC, and/or a neutrophil-dense tumor stroma may be less likely to achieve complete response using the standard of care chemoradiation regimen, and may benefit from the addition of a subsequent line of therapy.

Purpose: In advanced stage head and neck squamous cell cancers (HNSCC), approximately half of the patients with lymph node metastases (LNM) are not cured. Given the heterogeneous outcomes in these patients, we profiled the expression patterns of LNMs to identify the biological factors associated with patient outcomes. Experimental Design: We performed mRNAseq and miRNAseq on 72 LNMs and 29 matched primary tumors from 34 patients with HNSCC. Clustering identified molecular subtypes in LNMs and in primary tumors. Prediction Analysis of Microarrays algorithm identified a 73-gene classifier that distinguished LNM subtypes. Gene-set enrichment analysis identified pathways upregulated in LNM subtypes. Results: Integrative clustering identified three distinct LNM subtypes: (i) an immune subtype (Group 1), (ii) an invasive subtype (Group 2), and (iii) a metabolic/proliferative subtype (Group 3). Group 2 subtype was associated with significantly worse locoregional control and survival. LNM-specific subtypes were not observed in matched primary tumor specimens. In HNSCCs, breast cancers, and melanomas, a 73-gene classifier identified similar Group 2 LNM subtypes that were associated with worse disease control and survival only when applied to lymph node sites, but not when applied to other primary tumors or metastatic sites. Similarly, previously proposed prognostic classifiers better distinguished patients with worse outcomes when applied to the transcriptional profiles of LNMs, but not the profiles of primary tumors. Conclusions: The transcriptional profiles of LNMs better predict outcomes than transcriptional profiles of primary tumors. The LNMs display site-specific subtypes associated with worse disease control and survival across multiple cancer types.

Purpose: Low-dose CT screening can reduce lung cancer–related mortality. However, CT screening has an FDR of nearly 96%. We sought to assess whether urine samples can be a source for DNA methylation–based detection of non–small cell lung cancer (NSCLC). Experimental Design: This nested case–control study of subjects with suspicious nodules on CT imaging obtained plasma and urine samples preoperatively. Cases (n = 74) had pathologic confirmation of NSCLC. Controls (n = 27) had a noncancer diagnosis. We detected promoter methylation in plasma and urine samples using methylation on beads and quantitative methylation–specific real-time PCR for cancer-specific genes (CDO1, TAC1, HOXA7, HOXA9, SOX17, and ZFP42). Results: DNA methylation at cancer-specific loci was detected in both plasma and urine, and was more frequent in patients with cancer compared with controls for all six genes in plasma and in CDO1, TAC1, HOXA9, and SOX17 in urine. Univariate and multivariate logistic regression analysis showed that methylation detection in each one of six genes in plasma and CDO1, TAC1, HOXA9, and SOX17 in urine were significantly associated with the diagnosis of NSCLC, independent of age, race, and smoking pack-years. When methylation was detected for three or more genes in both plasma and urine, the sensitivity and specificity for lung cancer diagnosis were 73% and 92%, respectively. Conclusions: DNA methylation–based biomarkers in plasma and urine could be useful as an adjunct to CT screening to guide decision-making regarding further invasive procedures in patients with pulmonary nodules.

Photodynamic therapy is a non-invasive method where light activates a photosensitizer bound to cancer cells, generating reactive oxygen species and resulting in cell death. This study assessed the oncolytic potential of photodynamic therapy, comparing European Medicines Agency and United States Food and Drug Administration-approved 5-aminolevulinic acid (5-ALA) to a metalloporphyrin, Pd(T4), against a highly invasive uveal melanoma cell line (C918) in two- and three-dimensional models in vitro. Epithelial monolayer studies displayed strong oncolytic effects (&gt, 70%) when utilizing Pd(T4) at a fraction of the concentration, and reduced pre-illumination time compared to 5-ALA post-405 nm irradiance. When analyzed at sub-optimal concentrations, application of Pd(T4) and 5-ALA with 405 nm displayed cumulative effects. Lethality from Pd(T4)-photodynamic therapy was maintained within a three-dimensional model, including the more resilient vasculogenic mimicry-forming cells, though at lower rates. At high concentrations, modality of cell death exhibited necrosis partially dependent on reactive oxygen species. However, sub-optimal concentrations of photosensitizer exhibited an apoptotic protein expression profile characterized by increased Bax/Bcl-2 ratio and endoplasmic stress-related proteins, along with downregulation of apoptotic inhibitors CIAP-1 and -2. Together, our results indicate Pd(T4) as a strong photosensitizer alone and in combination with 5-ALA against C918 cells.

INTRODUCTION AND OBJECTIVES:Prostate cancer remains one of the most common cancer diagnoses among men worldwide, and the risk stratification system between indolent and aggressive disease continues to evolve. Radical prostatectomy surgery (RP) is a common curative treatment option, but incontinence and erectile dysfunction are frequent side effects of the procedure. As 30-40% of low-grade patients are upgraded to higher grade at RP, many patients choose RP treatment even though they harbor indolent disease. We have previously established a serum microRNA signature that predicts prostate cancer aggressiveness in order to further stratify patients and potentially help guide treatment decisions. Our current goals are to 1) validate the serum microRNA signature in a larger cohort and 2) determine if this prognostic signature is further enriched in circulating serum exosomes. Our hypothesis is that circulating exosomes harbor an enriched microRNA prognostic signature due to selective secretion from the prostat...

Several approaches to the preservation of biological materials at ambient temperature and the relative impact on sample stability and degradation are reviewed, with a focus on nucleic acids. This appraisal is undertaken within the framework of biobank risk, quality management systems, and accreditation, with a view to assessing how best to apply ambient temperature sample storage to ensure stability, reduce costs, improve handling logistics, and increase the efficiency of biobank procedures.

Herpes simplex virus 1 (HSV-1)-mediated oncolytic therapy is an emerging cancer treatment modality with potential effectiveness against a variety of malignancies. To better understand the interaction of HSV-1 with neoplastic cells, we inoculated three-dimensional (3D) cultures of human uveal melanoma cells with HSV-1. 3D melanoma cultures were established by placing tumor cells on the surface of a Matrigel matrix, which was followed by the growth of tumor cells on the matrix surface and invasion of the Matrigel matrix by some tumor cells to form multicellular tumor spheroids within the matrix. When established 3D melanoma cultures were inoculated with HSV-1 by placing virus on the surface of cultures, virus infection caused extensive death of melanoma cells growing on the surface of the 3D matrix and significantly decreased the number of tumor cell spheroids within the matrix. However, HSV-1 infection did not lead to a complete destruction of tumor cells in the 3D cultures during a 17-day observation period and, surprisingly, HSV-1 infection promoted the growth of some melanoma cells within the matrix as determined by the significantly increased size of residual viable multicellular tumor spheroids in virus-inoculated 3D cultures at 17 days after virus inoculation. Acyclovir treatment inhibited HSV-1-induced tumor cell killing but did not block the virus infection-induced increase in spheroid size. These findings suggest that although HSV-1 oncolytic virotherapy may cause extensive tumor cell killing, it may also be associated with the unintended promotion of the growth of some tumor cells.IMPORTANCE Cancer cells are exposed to HSV-1 during oncolytic virotherapy with the intention of killing tumor cells. Our observations reported here suggest that potential dangers of HSV-1 oncolytic therapy include promotion of growth of some tumor cells. Furthermore, our findings raise the possibility that HSV-1 infection of neoplastic cells during natural infections or vaccinations may promote the growth of tumors. Our study indicates that HSV-1 infection of 3D tumor cell cultures provides an experimental platform in which mechanisms of HSV-1-mediated promotion of tumor cell growth can be effectively studied.

This technical report presents quality control (QC) assays that can be performed in order to qualify clinical biospecimens that have been biobanked for use in research. Some QC assays are specific to a disease area. Some QC assays are specific to a particular downstream analytical platform. When such a qualification is not possible, QC assays are presented that can be performed to stratify clinical biospecimens according to their biomolecular quality.

Selenium supplementation of the diets of rodents has consistently been shown to suppress mammary carcinogenesis and some, albeit not all, human epidemiological studies have indicated an inverse association between selenium and breast cancer risk. In order to better understand the role selenium plays in breast cancer, 30 samples of tumor tissue were obtained from women with breast cancer and analyzed for selenium concentration, the levels of several selenium-containing proteins and the levels of the MnSOD anti-oxidant protein. Polymorphisms within the genes for these same proteins were determined from DNA isolated from the tissue samples. There was a wide range of selenium in these tissues, ranging from 24 to 854ng/gm. The selenium levels in the tissues were correlated to the genotype of the SELENOP selenium carrier protein, but not to other proteins whose levels have been reported to be responsive to selenium availability, including GPX1, SELENOF and SBP1. There was an association between a polymorphism in the gene for MnSOD and the levels of the encoded protein. These studies were the first to examine the relationship between selenium levels, genotypes and protein levels in human tissues. Furthermore, the obtained data provide evidence for the need to obtain data about the effects of selenium in breast cancer by examining samples from that particular tissue type.

The introduction of three-dimensional (3D) tumor cultures has revolutionized anticancer drug research as these cultures allow for the study of drug resistance mechanisms that cannot be explored in traditional two dimensional (2D) monolayer cultures. Discoveries in the 3D tumor culture field suggest that individualized drug sensitivity testing of solid tumor specimens through the establishment and use of 3D tumor cell cultures following tissue collection will become a routine service offered by modern tissue repositories as they expand from their traditional research role to active participation in personalized medicine. Unfortunately, most information related to 3D tumor cultures comes from studies using established tumor cell lines rather than primary tumor cultures. However, accumulation of genetic aberrations in cancer cell lines occurs with increasing number of passages severely limiting their usefulness for personalized medicine. There is only very limited information available concerning technologies and standard operating procedures for the efficient and routine isolation and processing of primary tumor cells for the establishment of 3D tumor cultures from solid tumor specimens. The purpose of this work was to review experimental data from the literature that may provide relevant information concerning the isolation and processing of primary tumor cells for the establishment of 3D tumor cultures. Information reviewed here may help biorepositories in the development and standardization of technologies and standard operating procedures related to the use of 3D tumor cultures.

To better understand the pathogenesis of dementia, it is important to understand histopathologic changes in neurodegenerative diseases because they might highlight key aspects of the degenerative process. In this study, the nuclear diameter of neurons and oligodendrocytes in selected temporal lobe areas were determined in autopsy tissue sections from patients with Alzheimer's disease (AD), Lewy body dementia (LBD) and controls. Our morphometric studies targeted neurons in the CA4 region of the pyramidal cell layer of the hippocampus, neurons in the granular layer of the dentate gyrus and oligodendrocytes in parahippocampal white matter. Mean neuronal nuclear diameters were not different among the studied groups. However, our studies revealed a statistically significant reduction of mean oligodendrocyte nuclear diameter in AD and LBD relative to controls. The reduction of the mean nucleus diameter of oligodendrocytes in LBD was independent of the presence of associated AD pathology in LBD. These findings for the first time identify decreased oligodendrocyte nucleus diameter as a morphologic feature of AD and LBD and may lead to a better understanding of the role of oligodendrocytes in AD and LBD pathogenesis.

Nectin-1 is a cell adhesion molecule that plays a role in interneuronal synapse formation, in axonal guidance during development and possibly in neuron-glia interactions. To better understand axonal changes in MS, nectin-1 expression was determined by immunohistochemistry in normal adult human cerebral white matter (n = 4) and in six MS plaques (three active and three inactive). The intensity of axonal nectin-1 expression was scored on a scale of 0 to 4+. In normal adult cerebral white matter, axons showed weak nectin-1 expression with a score of 1.25 ± 0.50. Axonal nectin-1 expression was significantly stronger within both active (score = 3.33 ± 0.289, p = 0.001) and inactive (score = 2.16 ± 0.29, p = 0.038) MS plaques than in normal white matter. Axons in white matter adjacent to MS plaques showed nectin-1 expression (score = 1.5 ± 0.50) that was not statistically different from normal controls (p = 0.542). These findings raise the possibility that increased expression of nectin-1 in MS lesions plays a role in the pathogenesis of MS through participation in axonal responses to injury and mediation of altered neuron-glia interactions relevant to myelination.

To better understand melanoma resistance to herpes simplex virus type 1 (HSV-1)-mediated oncolysis, traditional two-dimensional (2D) cultures and extracellular matrix (ECM) containing three-dimensional (3D) cultures of OCM1 and C918 uveal melanoma cells were infected with an HSV-1 strain that expresses the green fluorescent protein (GFP) marker during replication. Although 2D cultures were completely destroyed within a few days of HSV-1 inoculation, viable GFP-negative tumor cells remained detectable in 3D cultures for several weeks. Tumor cells with increased resistance to HSV-1 included cells that formed vasculogenic mimicry patterns and multicellular spheroids and cells that invaded Matrigel individually. Mechanisms of tumor resistance against HSV-1 in the 3D environment included impaired virus spread in the ECM and ECM-mediated inhibition of viral replication after viral entry into tumor cells. Observations also suggested that HSV-1 established quiescent infection in some tumor cells present in multicellular spheroids and that this could revert to productive viral infection when the tumor growth pattern changed. These findings indicate that 3D tumor cell cultures can be used to identify distinct tumor cell populations with increased resistance to HSV-1 and to explore mechanisms of ECM-mediated tumor resistance to oncolytic virotherapy.

Herpes virus entry mediator (HVEM) plays a critical role in the regulation of inflammation through interaction with its natural ligands LIGHT and lymphotoxin alpha and also serves as one of the entry receptors of herpes simplex virus (HSV). The purpose of this study was to better understand the expression of HVEM in the cornea and trigeminal ganglia (TG), which are important targets of HSV infection.Immunohistochemistry was used to define HVEM expression in the cornea and TG of normal and HSV-1 infected mice euthanized 2 to 5 days or 7 months following corneal inoculation of virus.We found that HVEM is widely expressed in the normal corneal epithelium and endothelium, is weakly and focally expressed in the corneal stroma, and is expressed in a portion of neurons and non-neuronal cells in the TG. Acute HSV-1 keratitis and ganglionitis were associated with increased HVEM expression in the corneal epithelium and stroma and in neurons and non-neuronal cells of TG, and many inflammatory cells in these tissues also expressed HVEM. TG derived from mice 7 months after virus inoculation demonstrated latent HSV-1 infection that was associated with increased HVEM expression in neurons and non-neuronal cells relative to uninfected control tissues. Latent TG also contained focal infiltrates of mononuclear inflammatory cells, many of which expressed HVEM. Corneas derived from latently infected mice demonstrated chronic keratitis, with no evidence of virus replication or increased HVEM expression in the corneal epithelium, and inflammatory cells present showed only weak HVEM expression.HVEM is expressed in the cornea and TG and therefore may serve as an HSV entry receptor in these tissues. Furthermore, these findings raise the possibility that changes in HVEM expression following ocular HSV-1 infection can modulate HSV spread and infection-induced inflammation in the cornea and TG.

The histological detection of laminin-rich vasculogenic mimicry patterns in human primary uveal melanomas is associated with death from metastases. We therefore hypothesized that highly invasive uveal melanoma cells forming vasculogenic mimicry patterns after exposure to a laminin-rich three-dimensional microenvironment would differentially express genes associated with invasive and metastatic behavior. However, we discovered that genes associated with differentiation (GDF15 and ATF3) and suppression of proliferation (CDKNa1/p21) were up-regulated in highly invasive uveal melanoma cells forming vasculogenic mimicry patterns, and genes associated with promotion of invasive and metastatic behavior such as CD44, CCNE2 (cyclin E2), THBS1 (thrombospondin 1), and CSPG2 (chondroitin sulfate proteoglycan; versican) were down-regulated. After forming vasculogenic mimicry patterns, uveal melanoma cells invaded only short distances, failed to replicate, and changed morphologically from the invasive epithelioid to the indolent spindle A phenotype. In human tissue samples, uveal melanoma cells within vasculogenic mimicry patterns assumed the spindle A morphology, and the expression of Ki67 was significantly reduced in adjacent melanoma cells. Thus, the generation of vasculogenic mimicry patterns is accompanied by dampening of the invasive and metastatic uveal melanoma genotype and phenotype and underscores the plasticity of these cells in response to cues from the microenvironment.

Given that expression of many genes changes when cells become malignant or are placed in different microenvironments, we asked whether these changes were accompanied by global reorganization of chromatin. We reasoned that sequestration or exposure of chromatin-sensitive sites to restriction enzymes could be used to detect this reorganization. We found that AluI-sensitive sites of nonmalignant cells were relatively more exposed compared to their malignant counterparts in cultured cells and human tumor samples. Changes in exposure and sequestration of AluI-sensitive sites in normal fibroblasts versus fibrosarcoma or those transfected with oncogenes, nonmalignant breast cells versus carcinomas and poorly metastatic versus highly invasive melanoma were shown to be independent of the cell cycle and may be influenced by proteins rich in disulfide bonds. Remarkably, regardless of degree of malignancy, AluI-sensitive sites became profoundly sequestered when cells were incubated with laminin, Matrigel, or a circular RGD peptide (RGD-C), but became exposed when cells were placed on collagen I or in serum-containing medium. Disruption of the actin cytoskeleton led to exposure, whereas disruption of microtubules or intermediate filaments exerted a sequestering effect. Thus, AluI-sensitive sites are more sequestered with increasing malignant behavior, but the sequestration and exposure of these sites is exquisitely sensitive to information conferred to the cell by molecules and biomechanical forces that regulate cellular and tissue architecture.

Microsomal triglyceride transfer protein (MTP) is essential for the assembly of apolipoprotein B-containing lipoproteins. Within the endoplasmic reticulum, it transfers lipid from the membrane to the forming lipoprotein. Recent evidence suggests that it may also function within the Golgi apparatus. To address this hypothesis, we developed a polyclonal antibody to MTP and used it in a series of studies on mouse liver and McArdle-RH7777 (McA) cells. Western blot analysis demonstrated the presence of MTP within mouse hepatic-Golgi apparatus-rich fractions. In addition, in vitro lipid transfer assays demonstrated the presence of triglyceride transfer activity within the Golgi fractions. Immunohistochemical studies with mouse liver demonstrated the presence of MTP within all hepatocytes, but not in nonparenchymal cells. The subcellular location of MTP in McA cells was investigated using confocal microscopy. MTP colocalized with the trans-Golgi network (TGN) 38 and Golgi SNARE (soluble N-ethylmalemide-sensitive factor attachment protein receptor) of 28 kDa (GS28), markers for the trans- and cis-Golgi apparatus, respectively. Morphometric analyses indicated that approximately 17% of the MTP signal colocalized with the TGN38, while 33% of the trans-Golgi marker colocalized with the MTP. Approximately 17% of the MTP signal colocalized with the GS28, whereas 53% of the cis-Golgi marker colocalized with the MTP. The results provide unequivocal evidence for the location of MTP within the Golgi apparatus, and further highlight the importance of this organelle in the assembly of lipoproteins.

Background: Despite the more recent advances, there is still no effective systemic therapy for Uveal Melanoma (UM). However, a better understanding of the role of oxidative stress in cancer, has more recently led to a completely new approach to the systemic therapy of cancer, and modulators of the oxidative balance, such as sodium ascorbate (ASC) or arsenic trioxide (ATO), have already entered advanced phases of preclinical and clinical development. Since high doses of ASC have already demonstrated a strong cytotoxic effect on different human cancer cell lines, we have undertaken the present investigation in order to test the sensitivity of OCM1 and C918 uveal melanoma (UM) cell lines to high doses of ASC, in vitro, as compared to ATO, a pro-oxidant drug which has already undergone extensive in vitro and pre-clinical investigation in UM. Methods: Both OCM1 and C918 UM cell lines have been exposed to increasing doses of either ASC or ATO, to build a dose-response curve around the peak plasma concentrations reached by both chemicals. The assessment of cell count and viability was performed with flow cytometry. Results: Both OCM1 and C918 UM cell lines are highly sensitive to ASC in the range of millimolar (mM) concentrations which can be usually reached by the intravenous injection of high doses of the compound. ATO at the dosages used in this study, never reached the LC50. When the exposure to ASC was reduced to two hours, it still had significant effects on survival of both OCM1 and C918 UM cells. Conclusions: This report shows that ASC is highly cytotoxic for both OCM1 and C918 UM cells, when used in high, pro-oxidant doses. To our knowledge, this is the first report showing that UM cells can be efficiently killed, in vitro, with high doses of ASC.

In previous studies we found that uveal melanoma cells grown in extracellular matrix (ECM)-containing three-dimensional (3D) cultures have increased resistance against herpes simplex virus type 1 (HSV-1)-mediated destruction relative to cells cultured without ECM. Using additional tumor cell types including MB-231 human breast cancer cells, PC-3 human prostate cancer cells, and P19 mouse embryonal carcinoma cells, we show here that tumor cell lines other than melanoma are also more resistant to HSV-1-mediated destruction in 3D cultures than cells grown in 2D. We also demonstrate here that one mechanism responsible for the increased resistance of tumor cells to HSV-1 infection in 3D cultures is an ECM-mediated inhibition of virus replication following virus entry into cells. These findings confirm and extend previous observations related to the role of the ECM in tumor resistance against HSV-1 and may lead to improved strategies of oncolytic virotherapy.

Purpose Cancer stem cells have increased resistance against a variety of anti-tumor treatment modalities. Vasculogenic mimicry (VM) patterns are present in numerous malignant tumor types, represent the formation of perfusion pathways by tumor cells, and their presence in tumors is associated with adverse outcome. Earlier we have shown that VM-forming tumor cells in three-dimensional (3D) uveal melanoma cultures have increased resistance against cytotoxic agents and oncolytic herpes simplex virus-mediated destruction. The purpose of the current study was to explore the possibility that this increased resistance of VM-forming tumor cells is due to a cancer stem cell phenotype. Methods The expression of cancer stem cell marker cluster of differentiation 271 (CD271) was determined in traditional two-dimensional (2D) and 3D cultures of C918 uveal melanoma cells by fluorescent immunocytochemistry. Results We found that the VM-forming tumor cell subpopulation in 3D cultures expressed CD271. In contrast, cells grown in 2D cultures and tumor cell subpopulations not participating in VM formation in 3D cultures were negative for CD271. Conclusions These findings suggest that VM-forming uveal melanoma cells acquire a cancer stem cell-like phenotype that may play a role in the increased therapy resistance of these cells.

Vasculogenic mimicry (VM) patterns are present in a wide variety of malignant tumors, represent the formation of perfusion pathways by tumor cells, and their presence in tumors is associated with adverse outcome (Maniotis et al., 1999; Folberg et al., 2000; Hendrix et al., 2003; Folberg and Maniotis, 2004; Dome et al., 2007). Mechanisms by which VM may contribute to adverse outcome are not well understood. Previous observations in our laboratory indicated that VM-forming tumor cells have increased resistance to herpes simplex virus-mediated oncolysis in three-dimensional (3D) uveal melanoma cultures (Valyi-Nagy et al., 2010). To determine whether VM-forming tumor cell subpopulations also have increased resistance against cytotoxic drugs, traditional two-dimensional (2D) and extracellular matrix (ECM, Matrigel)-containing 3D cultures of C918 uveal melanoma cells were established. In 2D cultures, C918 cells grew in monolayers. In 3D cultures, C918 cells formed a number of morphologically distinct tumor cell subpopulations that included cells that grew in monolayers on the Matrigel surface, cells that formed VM patterns, and cells that formed monolayers on the bottom of the culture dish. Following exposure to cisplatin or cadmium chloride, VM-forming tumor cells demonstrated prolonged survival relative to other tumor cell subpopulations in 3D cultures and cells grown in 2D. As presented in detail below, these findings suggest that increased drug resistance is a mechanism by which VMforming tumor cells contribute to adverse outcome.

Numerous epidemiological studies have proposed a link between herpes simplex virus (HSV) infection and several common chronic neuropsychiatric and neurodegenerative diseases. Experimental HSV infection of mice can lead to chronic behavioral and neurological deficits and chronic pain. While neuron injury and loss are well-documented consequences of the acute phase of infection, the pathologic consequences of latent HSV infection are poorly understood. To determine whether latent HSV infection can cause neuronal injury in mice, trigeminal ganglia (TG) derived from adult BALB/c mice 1, 12 and 31 weeks after corneal HSV type 1 (HSV-1) inoculation were analyzed for evidence of productive or latent HSV-1 infection, inflammation and changes in neuron size, density and number. We found that latent HSV-1 infection between 12 and 31 weeks after corneal virus inoculation was associated with inflammation and progressive deficits in mean neuron diameter, neuronal nucleus diameter, neuron density and neuron number in the TG relative to mock-infected controls. The extent of neuronal injury during latent infection correlated with the extent of inflammation. These studies demonstrate that latent HSV infection is associated with progressive neuronal pathology and may lead to a better understanding of the role of HSV infections in chronic neurological diseases.

Authentication of cell lines in biomedical research has been elevated to a very high priority. From a review of the literature, Lacroix1 reviewed the issue of cross-contamination of cell lines including the well known contamination of cell lines with HeLa cells,2 and the mis-identification of the ECV304 cell line as “immortalized endothelial cells” when these cells in fact originated from T24 bladder carcinoma cells.3 Lacroix 1 estimated that between 18 and 36% of cell lines have been misclassified. One survey at a large research institution suggested that fewer than 50% of researchers authenticate their cell lines.4 Nardone5 proposed recently that identification of cell lines be required of investigators before grants are awarded, and the National Institutes of Health subsequently called for researchers to authenticate cell lines as a prerequisite for grant funding.6

To determine whether herpes simplex virus type 1 (HSV-1) infection causes oxidative stress and lipid peroxidation in cultured neural cells, mouse P19 embryonal carcinoma cells were differentiated into cells with neural phenotypes (P19N cells) by retinoic acid and were then infected with HSV-1. Cellular levels of reactive oxygen species (ROS) and the release of lipid peroxidation by-products into the tissue culture medium were then measured by the generation of fluorescent markers hydroxyphenyl fluorescein and a stable chromophore produced by lipid peroxidation products, malondialdehyde (MDA) and hydroxyalkenals (4-HAEs; predominantly 4-hydroxy-2-nonenal [HNE]), respectively. HSV-1 infection increased ROS levels in neural cells as early as 1 h post infection (p.i.) and ROS levels remained elevated at 24 h p.i. This viral effect required viral entry and replication as heat- and ultraviolet light-inactivated HSV-1 were ineffective. HSV-1 infection also was associated with increased levels of MDA/HAE in the culture medium at 2 and 4 h p.i., but MDA/HAE levels were not different from those detected in mock infected control cultures at 1, 6, and 24 h p.i. HSV-1 replication in P19N cells was inhibited by the antioxidant compound ebselen and high concentrations of HNE added to the cultures, but was increased by low concentrations of HNE. These findings indicate that HSV-1 infection of neural cells causes oxidative stress that is required for efficient viral replication. Furthermore, these observations raise the possibility that soluble, bioactive lipid peroxidation by-products generated in infected neural cells may be important regulators of HSV-1 pathogenesis in the nervous system.

The clinical tendency for uveal melanoma to spread first and preferentially to the liver is well documented.1 In developing animal models of uveal melanoma, most investigators have focused on studying the mechanisms by which tumors develop in the animal eye and generate hepatic metastases. Strategies for modeling primary uveal melanoma include the induction of tumors by feline retroviruses2 and chemical carcinogens,3,4 the development of transgenic models of primary uveal melanoma,5-10 and the transplantation of melanoma cell lines—animal11-15 and human16-18—to the animal eye. The literature summarizing animal models of uveal melanoma was reviewed recently.19 Models of uveal melanoma based on the implantation of tumor into the animal eye have been the focus of several innovative experimental approaches designed to reduce the spread of tumor from the eye to the liver.16,20-23 In these models, the entire metastatic cascade—dislodgement from the primary tumor, entrance to the microcirculation, extravasation, and proliferation within the liver—are all potential targets of experimental therapy. Certainly, the prevention of metastasis in high-risk patients is a clinical priority; however, after a patient develops metastatic uveal melanoma, the clinical focus shifts entirely to eradication of the established metastases. Although there has been considerable progress in developing vision-sparing alternatives to the removal of the eye for treatment of the primary tumor, there have been no significant improvements in the treatment of hepatic metastases. Little is known in the ophthalmic research community about the biological behavior of uveal melanoma once it has arrived in the liver. Since ophthalmic oncologic research has focused to date nearly exclusively on the entire metastatic cascade by introducing uveal melanoma into the eye of experimental animals, we modified a colon cancer model of direct injection24 to study the biology of uveal melanoma in the liver. We discovered that nodules of uveal melanoma develop in predictable time courses in the mouse liver, followed by secondary melanoma deposits in the lungs. Hepatic tumor nodules and secondary pulmonary lesions develop within 4 to 8 weeks after injection into the liver, depending on the invasive properties of the cell line injected. The histology of melanoma nodules in the liver that form after direct injection in mice is similar to the histology of human metastatic uveal melanoma in the liver. Similar to primary and metastatic human uveal melanomas, laminin-rich vasculogenic mimicry patterns formed in hepatic lesions, and these patterns conducted fluid. The direct-injection model can therefore be used to study biological events that are involved in establishing and maintaining foci of uveal melanoma in the liver, and this model provides an additional approach to the development of novel, effective, and nontoxic therapies targeted to the treatment of metastatic uveal melanoma.