Vanessa M. Morales-Tirado

The Toll-like receptor 5 agonist entolimod suppresses hepatic metastases in a murine model of ocular melanoma via an NK cell-dependent mechanism

Uveal melanoma (UM) is the most common primary cancer of the eye in adults and progresses to metastatic disease predominantly of the liver in ∼50% of patients. In these cases, life expectancy averages just 9 months due to the lack of effective treatment options. The Toll-like receptor 5 (TLR5) agonist entolimod (former name CBLB502) rapidly activates TLR5-NF-κB signaling in hepatocytes and suppresses growth of both TLR5-expressing and non-expressing tumors in the liver through mobilization and activation of innate and adaptive immune mechanisms. The goal of this study was to explore the potential of entolimod as an immunotherapeutic agent against hepatic metastasis of UM using the TLR5-positive B16LS9 mouse model of ocular melanoma. Mice were given seven subcutaneous injections of vehicle or entolimod given 72 h apart started one day before, on the same day or three days after intraocular injection of B16LS9 cells. All tested regimens of entolimod treatment resulted in significantly reduced B16LS9 metastasis to the liver. Entolimod induced mobilization of natural killer (NK) cells to the liver and stimulated their maturation, differentiation and activation. Antibody-mediated depletion of NK cells from mice abrogated entolimods antimetastatic activity in the liver and eliminated the entolimod-elicited in vitro cytotoxic activity of hepatic lymphocytes against B16LS9 cells. These results provide pre-clinical evidence of entolimods efficacy against hepatometastasis of UM and support its further development as an anticancer immunotherapeutic drug.

Inhibition of MMP-2 and MMP-9 decreases cellular migration, and angiogenesis in in vitro models of retinoblastoma

BackgroundRetinoblastoma (Rb) is the most common primary intraocular tumor in children. Local treatment of the intraocular disease is usually effective if diagnosed early; however advanced Rb can metastasize through routes that involve invasion of the choroid, sclera and optic nerve or more broadly via the ocular vasculature. Metastatic Rb patients have very high mortality rates. While current therapy for Rb is directed toward blocking tumor cell division and tumor growth, there are no specific treatments targeted to block Rb metastasis. Two such targets are matrix metalloproteinases-2 and -9 (MMP-2, −9), which degrade extracellular matrix as a prerequisite for cellular invasion and have been shown to be involved in other types of cancer metastasis. Cancer Clinical Trials with an anti-MMP-9 therapeutic antibody were recently initiated, prompting us to investigate the role of MMP-2, −9 in Rb metastasis.MethodsWe compare MMP-2, −9 activity in two well-studied Rb cell lines: Y79, which exhibits high metastatic potential and Weri-1, which has low metastatic potential. The effects of inhibitors of MMP-2 (ARP100) and MMP-9 (AG-L-66085) on migration, angiogenesis, and production of immunomodulatory cytokines were determined in both cell lines using qPCR, and ELISA. Cellular migration and potential for invasion were evaluated by the classic wound-healing assay and a Boyden Chamber assay.ResultsOur results showed that both inhibitors had differential effects on the two cell lines, significantly reducing migration in the metastatic Y79 cell line and greatly affecting the viability of Weri-1 cells. The MMP-9 inhibitor (MMP9I) AG-L-66085, diminished the Y79 angiogenic response. In Weri-1 cells, VEGF was significantly reduced and cell viability was decreased by both MMP-2 and MMP-9 inhibitors. Furthermore, inhibition of MMP-2 significantly reduced secretion of TGF-β1 in both Rb models.ConclusionsCollectively, our data indicates MMP-2 and MMP-9 drive metastatic pathways, including migration, viability and secretion of angiogenic factors in Rb cells. These two subtypes of matrix metalloproteinases represent new potential candidates for targeted anti-metastatic therapy for Rb.

Isolation and Molecular Profiling of Primary Mouse Retinal Ganglion Cells: Comparison of Phenotypes from Healthy and Glaucomatous Retinas

Loss of functional retinal ganglion cells (RGC) is an element of retinal degeneration that is poorly understood. This is in part due to the lack of a reliable and validated protocol for the isolation of primary RGCs. Here we optimize a feasible, reproducible, standardized flow cytometry-based protocol for the isolation and enrichment of homogeneous RGC with the Thy1.2hiCD48negCD15negCD57neg surface phenotype. A three-step validation process was performed by: (1) genomic profiling of 25-genes associated with retinal cells; (2) intracellular labeling of homogeneous sorted cells for the intracellular RGC-markers SNCG, brain-specific homeobox/POU domain protein 3A (BRN3A), TUJ1, and RNA-binding protein with multiple splicing (RBPMS); and (3) by applying the methodology on RGC from a mouse model with elevated intraocular pressure (IOP) and optic nerve damage. Use of primary RGC cultures will allow for future careful assessment of important cell specific pathways in RGC to provide mechanistic insights into the declining of visual acuity in aged populations and those suffering from retinal neurodegenerative diseases.

Pioglitazone restores IGFBP-3 levels through DNA PK in retinal endothelial cells cultured in hyperglycemic conditions

PURPOSE Previously, we reported that pioglitazone prevented insulin resistance and cell death in type 2 diabetic retina by reducing TNFα and suppressor of cytokine signaling 3 (SOCS3) levels. Numerous reports suggest prominent vasoprotective effects of insulin growth factor binding protein-3 (IGFBP-3) in diabetic retinopathy. We hypothesized that pioglitazone protects against retinal cell apoptosis by regulating IGFBP-3 levels, in addition to reducing TNFα. The current study explored potential IGFBP-3 regulatory pathways by pioglitazone in retinal endothelial cells cultured in high glucose. METHODS Primary human retinal endothelial cells (REC) were grown in normal (5 mM) and high glucose (25 mM) and treated with pioglitazone for 24 hours. Cell lysates were processed for Western blotting and ELISA analysis to evaluate IGFBP-3, TNFα, and cleaved caspase 3 protein levels. RESULTS Our results show that treatment with pioglitazone restored the high glucose-induced decrease in IGFBP-3 levels. This regulation was independent of TNFα actions, as reducing TNFα levels with siRNA did not prevent pioglitazone from increasing IGFBP-3 levels. Pioglitazone required protein kinase A (PKA) and DNA-dependent protein kinase (DNA PK) activity to regulate IGFBP-3, as specific inhibitors for each protein prevented pioglitazone-mediated normalization of IGFBP-3 in high glucose. Insulin growth factor binding protein-3 activity was increased and apoptosis decreased by pioglitazone, which was eliminated when serine site 156 of IGFBP-3 was mutated suggesting a key role of this phosphorylation site in pioglitazone actions. CONCLUSIONS Our findings suggest that pioglitazone mediates regulation of IGFBP-3 via activation of PKA/DNA PK pathway in hyperglycemic retinal endothelial cells.

Novel quinic acid derivative KZ-41 prevents retinal endothelial cell apoptosis without inhibiting retinoblastoma cell death through p38 signaling.

PURPOSE To determine whether a novel NF-κB inhibitor, KZ-41, can inhibit melphalans actions on retinal endothelial cell (REC) inflammation and apoptosis, without eliminating the chemotherapeutic efficacy of melphalan on cell death of retinoblastoma cells (Y79). METHODS RECs were cultured in M131 medium supplemented with growth factors and antibiotics. Once cells reached confluence, they were treated with or without 10 μM KZ-41, following treatment with 4 μg/mL melphalan. Cell proteins were extracted and analyzed for intracellular adhesion molecule 1 (ICAM-1) levels and Cell Death ELISA. RECs were also transfected with or without NF-κB siRNA or treated with SB202190 (p38 [mitogen activated protein kinase] MAPK inhibitor) before melphalan treatment to determine the involvement of NF-κB and p38 MAPK in REC apoptosis and ICAM-1 levels. We also cultured retinoblastoma cells (Y79) in RMPI-1640 medium supplemented with 20% fetal bovine serum and performed a Cell Death ELISA after melphalan + KZ-41 treatment to determine if the treatments altered melphalans ability to promote cell death of Y79 cells. RESULTS KZ-41 inhibited melphalan-stimulation of ICAM-1 levels and REC apoptosis, whereas KZ-41 did not alter melphalans effects on Y79 cells. KZ-41s protective effects on REC were mediated through p38 MAPK activation. Although KZ-41 blocked both NF-κB- and p38 MAPK-dependent ICAM-1 stimulation; the p38 MAPK/ICAM-1 pathway appears to be the primary pathway involved in melphalan-induced REC apoptosis. CONCLUSIONS KZ-41 protects REC against melphalan-induced upregulation of ICAM-1 and apoptosis through p38 MAPK-dependent pathways.

Multipronged approach to identify and validate a novel upstream regulator of Sncg in mouse retinal ganglion cells.

Loss of retinal ganglion cells (RGCs) is one of the hallmarks of retinal neurodegenerative diseases, glaucoma being one of the most common. Mechanistic studies on RGCs are hindered by the lack of sufficient primary cells and consensus regarding their signature markers. Recently, γ‐synuclein (SNCG) has been shown to be highly expressed in the somas and axons of RGCs. In various mouse models of glaucoma, downregulation of Sncg gene expression correlates with RGC loss. To investigate the role of Sncg in RGCs, we used a novel systems genetics approach to identify a gene that modulates Sncg expression, followed by confirmatory studies in both healthy and diseased retinae. We found that chromosome 1 harbors an expression quantitative trait locus that modulates Sncg expression in the mouse retina, and identified the prefoldin‐2 (PFDN2) gene as the candidate upstream modulator of Sncg expression. Our immunohistochemical analyses revealed similar expression patterns in both mouse and human healthy retinae, with PFDN2 colocalizing with SNCG in RGCs and their axons. In contrast, in retinae from glaucoma subjects, SNCG levels were significantly reduced, although PFDN2 levels were maintained. Using a novel flow cytometry‐based RGC isolation method, we obtained viable populations of murine RGCs. Knocking down Pfdn2 expression in primary murine RGCs significantly reduced Sncg expression, confirming that Pfdn2 regulates Sncg expression in murine RGCs. Gene Ontology analysis indicated shared mitochondrial function associated with Sncg and Pfdn2. These data solidify the relationship between Sncg and Pfdn2 in RGCs, and provide a novel mechanism for maintaining RGC health.

Isolation of primary murine retinal ganglion cells (RGCs) by flow cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Pioglitazone alters monocyte populations and stimulates recent thymic emigrants in the BBDZR/Wor type 2 diabetes rat model.

BackgroundType 2 diabetes is commonly characterized by insulin deficiency and decreased sensitivity of insulin receptors, leading to a chronic state of hyperglycemia in individuals. Disease progression induces changes in the immune profile that engenders a chronic inflammatory condition. Thiazolidinedione (TDZ) drugs, such as Pioglitazone (Pio), aid in controlling disease symptoms. While the mechanisms by which Pio controls hyperglycemia are beginning to be understood, relatively little is known about the effects of Pio on suppression of the systemic immune phenotype, attributed to visceral adipose tissue and macrophages.MethodsHere, we utilize the recently developed BBDZR/Wor type 2 diabetes rat model to test our hypothesis that a selective in vivo growth of CD3+T cells in the spleen contributes to the increase in T lymphocytes, including Tregs, independent of visceral adipose tissue. We investigated the systemic effects of Pio on multifactorial aspects of the disease-induced immune phenotype both in vivo and in vitro in normal, non-diabetic animals and in disease.ResultsOur work revealed that Pio reversed the lymphopenic status of diabetic rats, in part by an increase in CD3+ T lymphocytes and related subsets. Moreover, we found evidence that Pio caused a selective growth of newly differentiated T lymphocytes, based on the presence of recent thymic emigrants in vivo. To investigate effects of Pio on the inflammatory milieu, we examined the production of the signature cytokines TNF-α and IL-1β and found they were reduced by Pio-treatment, while the levels of IL-4, an anti-inflammatory mediator, were significantly increased in a Pio-dependent manner. The increase in IL-4 production, although historically attributed to macrophages from visceral adipose tissue under other conditions, came also from CD3+ T lymphocytes from the spleen, suggesting splenocytes contribute to the Pio-induced shift towards an anti-inflammatory phenotype.ConclusionsWe show for the first time that Pio treatment significantly suppresses the systemic inflammatory status in the BBDZR/Wor type 2 diabetes rat model by the selective growth of newly differentiated CD3+ T cells and by increasing CD3+IL-4 production in immigrant spleen lymphocytes.

Targeting the Platelet-Derived Growth Factor-beta Stimulatory Circuitry to Control Retinoblastoma Seeds

Purpose Vitreous seeding remains the primary reason for treatment failure in eyes with retinoblastoma (Rb). Systemic and intra-arterial chemotherapy, each with its own inherent set of complications, have improved salvage rates for eyes with advanced disease, but the location and biology of vitreous seeds present a fundamental challenge in developing treatments with minimal toxicity and risk. The aim of this study was to target the platelet-derived growth factor (PDGF)– PDGF-receptor β (PDGFRβ) signaling pathway and investigate its role in the growth of Rb seeds, apoptotic activity, and invasive potential. Methods We performed ex vivo analyses on vitreous samples from Rb patients that underwent enucleation and from patient-derived xenografts. These samples were evaluated by quantitative PCR, immunohistochemistry, and ELISA. The effects of disruption of the PDGF-PDGFRβ signaling pathway, both by pharmacologic and genomic knockdown approaches, were evaluated in vitro by cell proliferation and apoptotic assays, quantitative PCR analyses, Western blotting, flow cytometry, and imaging flow cytometry. A three-dimensional cell culture system was generated for in-depth study of Rb seeds. Results Our results demonstrated that PDGFRβ signaling is active in the vitreous of Rb patients and patient-derived xenografts, sustaining growth and survival in an AKT-, MDM2-, and NF-κB-dependent manner. The novel three-dimensional cell culture system mimics Rb seeds, as the in vitro generated spheroids have similar morphologic features to Rb seeds and mimicked their natural physiology. Conclusions Targeting the PDGFRβ pathway in vitro reduces Rb cell growth, survival, and invasiveness and could augment current therapies. This represents a novel signaling pathway for potential targeted therapy to further improve ocular survival in advanced Rb.

Uveal metastases in the mid-southeastern United States: a single-institution experience

Purpose To report the clinical features of uveal metastases in a geographic region associated with high tobacco use. Methods Medical records from all patients diagnosed with uveal metastasis at a single tertiary referral center between 2000 and 2017 were retrospectively reviewed. The clinical features and the primary tumor site associated with each metastatic lesion were recorded. Results Ninety-nine uveal metastatic tumors were identified in 85 eyes of 74 patients (34 males). Median age at diagnosis was 62 years. Median tumor diameter was 11.6 mm and median height was 3.1 mm. Carcinoma of the lung was the most common primary tumor occurring in 37 patients (50%) followed by breast in 16 patients (21%). Among females, metastatic lesions originated from the lung in 18 patients and from the breast in 16 patients. Median survival following intraocular metastasis was 9 months for patients with a primary lung malignancy and 36 months for patients with breast cancer (log-rank test, P=0.002). Conclusion Intraocular metastasis is more frequently observed in patients with carcinomas of the lung rather than breast at our treatment center. Both regional and global changes in cancer epidemiology most likely account for the findings in this study.