Retinoblastoma (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.
We 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.
Our 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.
Collectively, 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.
Metastases account for 90% of all cancer-related deaths, becoming a therapeutic problem. Approximately 50% of all uveal melanoma (UM) patients will develop metastases, mainly in the liver. ...Post-mortem analyses of livers from metastatic UM patients showed two different metastatic growth patterns: infiltrative and nodular. The infiltrative pattern exhibits tumor infiltration directly to the hepatic lobule and minimal angiogenesis. The nodular pattern shows clusters of tumor cells around the portal venules that efface the liver parenchyma. We recently demonstrated Natural Killer (NK) cells play a pivotal role in the control of hepatic metastases and the pigment epithelial-derived factor (PEDF) controls angiogenesis in the liver using our established ocular melanoma animal model. In this study we investigated the role of NK cells and PEDF in the development of metastatic growth patterns, as this can contribute to the development of novel therapeutics specific towards each growth pattern.
We utilize our established ocular melanoma animal model by inoculation of B16-LS9 melanoma cells into C57BL/6 J mice (WT), anti-asialo GM1-treated C57BL/6 J mice (NK-depleted), and PEDF
C57BL/6 J mice. Three weeks after inoculation we evaluated the metastatic growth patterns and stratified them based of the numbers of tumor cells. To evaluate angiogenesis the mean vascular density (MVD) was calculated. The immune compartment of the liver was analyzed by flow cytometry.
Our in vivo work showed two distinct metastatic growth patterns, the infiltrative and nodular, recapitulating the post-mortem analyses on human liver tissue. We discovered NK cells control the infiltrative growth. In contrast, PEDF controlled anti-angiogenic responses, showing higher MVD values compared to NK-depleted and WT animals. The myeloid lineage, comprised of monocytes, macrophages, and myeloid-derived suppressor cells, was reduced in the absence of NK cells or PEDF.
Our animal model recapitulates the metastatic growth patterns observed in the human disease. We demonstrated a role for NK cells in the development of the infiltrative growth pattern, and a role for PEDF in the development of the nodular pattern. The understanding of the complexity associated with the metastatic progression has profound clinical implications in the diagnostic and disease-management as we can develop and direct more effective therapies.
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.
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.
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.
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.
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.
Abstract FOXP3 is critical for the development and function of CD4+ CD25bright natural regulatory T cells (nTreg). Individuals harboring mutations in FOXP3 develop immune dysregulation, ...polyendocrinopathy, enteropathy, X-linked syndrome (IPEX). We describe a child diagnosed with IPEX who underwent a reduced intensity, T and B cell depleted, matched unrelated donor bone marrow transplant followed by clinical resolution. Using lineage-specific donor chimerism studies, we demonstrate that non-myeloablative HSCT resolves disease in the context of low level donor hematopoietic stem cell (HSC) engraftment. Despite low-levels of donor HSC, thymically-derived nTreg and to a lesser extent CD4+ and CD8+ T cells, exhibit a selective in vivo growth advantage for populations containing a functional FOXP3 gene. Moreover, nTreg from this patient show regulatory function directly ex vivo. These results have implications for improving clinical therapy for patients with IPEX and provide mechanistic insight into the in vivo development of human nTreg and unexpectedly, non-regulatory T cells.
Abstract Purpose Report the outcomes of survival, local control, visual acuity and eye retention in patients treated with repeat episcleral plaque brachytherapy (EPBT) for locally recurrent posterior ...uveal melanoma (PUM). Design Retrospective, interventional case series Methods Setting: Institutional Patient Population: 1201 patients that underwent Iodine-125 (I-125) EPBT as primary treatment for PUM between 1985 and 2015 Inclusion criteria: Development of locally recurrent disease and retreatment with I-125 EPBT Observation Procedures: Clinical records review Main Outcome Measures: Visual acuity, Kaplan-Meier estimates of survival, local control, metastasis and loss of the eye over the duration of follow up Results Twenty-seven patients (13 men) met our inclusion criteria. Median follow up from initial treatment was 100 months (14–365 months), while median time-to-local recurrence was 43 months (9–185 months). Median follow up after retreatment was 47 months (3–120 months). Kaplan-Meier estimate for local control at 5 years was 77.2% (95% confidence interval CI, 53.29-89.91%). All marginal recurrences were successfully retreated while 6 of 15 patients with central recurrence developed subsequent re-recurrence following salvage EPBT. Median visual acuity was 20/70 (20/20–Counting Fingers at 1 foot) at time of recurrence and declined to Counting Fingers (20/25–Hand Motion) at the most recent follow-up exam. Kaplan-Meier estimate for absence of metastatic disease at 5 years was 78.5% (95% CI, 54.77-90.70%). Conclusions Repeat I-125 EPBT offers a viable alternative to enucleation in patients with local recurrence of PUM yielding high rates of local control with predictable decline in visual acuity.
To report the clinical features of uveal metastases in a geographic region associated with high tobacco use.
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.
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,
=0.002).
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.
Background/Aims: The aim of this study is to report the burden of ocular morbidity following iodine-125 episcleral plaque brachytherapy (EPBT) in the treatment of American Joint Committee on Cancer ...(AJCC) T4-staged posterior uveal melanoma (PUM). Methods: Clinical records of patients with T4-staged PUM treated with 125 I EPBT were analyzed for incidence of treatment failure and radiation-induced complications. Results: Cumulative incidence of local treatment failure was 9% (95% CI 5–15%) at 5 years and was associated with decreased tumor height (HR = 0.78; p = 0.01). Cumulative incidence of enucleation at 5 years was 21% and was correlated with worsening baseline visual acuity (HR = 1.42; p = 0.05). Increasing patient age was associated with higher rates of vitreous hemorrhage (HR = 1.03; p = 0.02) and cataract surgery (HR = 1.05; p < 0.001). Increased tumor height was associated with higher rates of neovascular glaucoma (HR = 1.16; p = 0.03) and vitreous hemorrhage (HR = 1.23; p < 0.001). Conclusion: 125 I EPBT is an effective treatment for T4-staged PUM and achieves high rates of local control. Treatment failure appears to be more common among minimally elevated tumors. Other causes of ocular morbidity were associated with increasing tumor height, patient age, and baseline visual acuity.
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 Thy1.2(hi)CD48(neg)CD15(neg)CD57(neg) 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.
Liver metastases often progress from primary cancers including uveal melanoma (UM), breast, and colon cancer. Molecular biomarker imaging is a new non-invasive approach for detecting early stage ...tumors. Here, we report the elevated expression of chemokine receptor 4 (CXCR4) in liver metastases in UM patients and metastatic UM mouse models, and development of a CXCR4-targeted MRI contrast agent, ProCA32.CXCR4, for sensitive MRI detection of UM liver metastases. ProCA32.CXCR4 exhibits high relaxivities (
= 30.9 mM
s
,
= 43.2 mM
s
, 1.5 T;
= 23.5 mM
s
,
= 98.6 mM
s
, 7.0 T), strong CXCR4 binding (
= 1.10 ± 0.18 μM), CXCR4 molecular imaging capability in metastatic and intrahepatic xenotransplantation UM mouse models. ProCA32.CXCR4 enables detecting UM liver metastases as small as 0.1 mm
. Further development of the CXCR4-targeted imaging agent should have strong translation potential for early detection, surveillance, and treatment stratification of liver metastases patients.
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