Exploration of Imaging Biomarkers for Metabolically-Targeted Osteosarcoma Therapy in a Murine Xenograft Model.

Background: Osteosarcoma (OS) is an aggressive pediatric cancer with unmet therapeutic needs. Glutaminase 1 (GLS1) inhibition, alone and in combination with metformin, disrupts the bioenergetic demands of tumor progression and metastasis, showing promise for clinical translation. Materials and Methods: Three positron emission tomography (PET) clinical imaging agents, [18F]fluoro-2-deoxy-2-D-glucose ([18F]FDG), 3'-[18F]fluoro-3'-deoxythymidine ([18F]FLT), and (2S, 4R)-4-[18F]fluoroglutamine ([18F]GLN), were evaluated in the MG63.3 human OS xenograft mouse model, as companion imaging biomarkers after treatment for 7 d with a selective GLS1 inhibitor (CB-839, telaglenastat) and metformin, alone and in combination. Imaging and biodistribution data were collected from tumors and reference tissues before and after treatment. Results: Drug treatment altered tumor uptake of all three PET agents. Relative [18F]FDG uptake decreased significantly after telaglenastat treatment, but not within control and metformin-only groups. [18F]FLT tumor uptake appears to be negatively affected by tumor size. Evidence of a flare effect was seen with [18F]FLT imaging after treatment. Telaglenastat had a broad influence on [18F]GLN uptake in tumor and normal tissues. Conclusions: Image-based tumor volume quantification is recommended for this paratibial tumor model. The performance of [18F]FLT and [18F]GLN was affected by tumor size. [18F]FDG may be useful in detecting telaglenastat's impact on glycolysis. Exploration of kinetic tracer uptake protocols is needed to define clinically relevant patterns of [18F]GLN uptake in patients receiving telaglenastat.

Adjuvant Sirolimus Does Not Improve Outcome in Pet Dogs Receiving Standard-of-Care Therapy for Appendicular Osteosarcoma: A Prospective, Randomized Trial of 324 Dogs.

PURPOSE: The mTOR pathway has been identified as a key nutrient signaling hub that participates in metastatic progression of high-grade osteosarcoma. Inhibition of mTOR signaling is biologically achievable with sirolimus, and might slow the outgrowth of distant metastases. In this study, pet dogs with appendicular osteosarcoma were leveraged as high-value biologic models for pediatric osteosarcoma, to assess mTOR inhibition as a therapeutic strategy for attenuating metastatic disease progression. PATIENTS AND METHODS: A total of 324 pet dogs diagnosed with treatment-naïve appendicular osteosarcoma were randomized into a two-arm, multicenter, parallel superiority trial whereby dogs received amputation of the affected limb, followed by adjuvant carboplatin chemotherapy ± oral sirolimus therapy. The primary outcome measure was disease-free interval (DFI), as assessed by serial physical and radiologic detection of emergent macroscopic metastases; secondary outcomes included overall 1- and 2-year survival rates, and sirolimus pharmacokinetic variables and their correlative relationship to adverse events and clinical outcomes. RESULTS: There was no significant difference in the median DFI or overall survival between the two arms of this trial; the median DFI and survival for standard-of-care (SOC; defined as amputation and carboplatin therapy) dogs was 180 days [95% confidence interval (CI), 144-237] and 282 days (95% CI, 224-383) and for SOC + sirolimus dogs, it was 204 days (95% CI, 157-217) and 280 days (95% CI, 252-332), respectively. CONCLUSIONS: In a population of pet dogs nongenomically segmented for predicted mTOR inhibition response, sequentially administered adjuvant sirolimus, although well tolerated when added to a backbone of therapy, did not extend DFI or survival in dogs with appendicular osteosarcoma.

Proinflammatory cytokines decrease the expression of genes critical for RPE function.

PURPOSE: Proinflammatory cytokines interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-1 beta (IL-1ß) secreted by infiltrating lymphocytes or macrophages may play a role in triggering RPE dysfunction associated with age-related macular degeneration (AMD). Binding of these proinflammatory cytokines to their specific receptors residing on the RPE cell surface can activate signaling pathways that, in turn, may dysregulate cellular gene expression. The purpose of the present study was to investigate whether IFN-γ, TNF-α, and IL-1ß have an adverse effect on the expression of genes essential for RPE function, employing the RPE cell line ARPE-19 as a model system. METHODS: ARPE-19 cells were cultured for 3-4 months until they exhibited epithelial morphology and expressed mRNAs for visual cycle genes. The differentiated cells were treated with IFN-γ, TNF-α, and/or IL-1ß, and gene expression was analyzed with real-time PCR analysis. Western immunoblotting was employed for the detection of proteins. RESULTS: Proinflammatory cytokines (IFN-γ + TNF-α + IL-1ß) greatly increased the expression of chemokines and cytokines in cultured ARPE-19 cells that exhibited RPE characteristics. However, this response was accompanied by markedly decreased expression of genes important for RPE function, such as CDH1, RPE65, RDH5, RDH10, TYR, and MERTK. This was associated with decreased expression of the genes MITF, TRPM1, and TRPM3, as well as microRNAs miR-204 and miR-211, which are known to regulate RPE-specific gene expression. The decreased expression of the epithelial marker gene CDH1 was associated with increased expression of mesenchymal marker genes (CDH2, VIM, and CCND1) and epithelial-mesenchymal transition (EMT) promoting transcription factor genes (ZEB1 and SNAI1). CONCLUSIONS: RPE cells exposed to proinflammatory cytokines IFN-γ, TNF-α, and IL-1ß showed decreased expression of key genes involved in the visual cycle, epithelial morphology, and phagocytosis. This adverse effect of proinflammatory cytokines, which could be secreted by infiltrating lymphocytes or macrophages, on the expression of genes indispensable for RPE function may contribute to the RPE dysfunction implicated in AMD pathology.

Inflammatory cytokines regulate secretion of VEGF and chemokines by human conjunctival fibroblasts: Role in dysfunctional tear syndrome.

Ocular surface inflammation is one of the primary mechanisms associated with dysfunctional tear syndrome (DTS), also known as dry eye disease. DTS, more prevalent in older populations, causes ocular discomfort and visual disturbance due to dryness on the surface layer in the eye. We used human conjunctival fibroblast cultures (HCJVF) to investigate the effects of inflammatory cytokines IFN-γ, TNF-α and IL-1ß (ITI) on the secretions of VEGF and chemokines. Our results demonstrate the elevated secretion of angiogenic VEGF molecules by ITI without affecting anti-angiogenic molecules, PEDF, endostatin, thrombospondin and sVEGF-R1. The secretion of interferon-γ inducible chemokines, CXCL9, -10, -11 by HCJVF were significantly enhanced by ITI. Our in vitro study supports previously reported observations of elevated VEGF and chemokines in tear fluids of DTS patients, reiterating the role of inflammatory reactions in DTS.

Resveratrol attenuates CXCL11 expression induced by proinflammatory cytokines in retinal pigment epithelial cells.

Dysfunction of the retinal pigment epithelium (RPE) resulting from chronic inflammation is implicated in the pathogenesis of age-related macular degeneration (AMD). RPE cells adjacent to drusen deposits in the AMD eye are known to contain CXCL11, a chemokine involved in inflammatory cell recruitment. We investigated the CXCL11 production by the human RPE (ARPE-19) cells under inflammatory conditions and tested its response to resveratrol, a naturally occurring anti-inflammatory antioxidant. A proinflammatory cytokine mixture consisting of IFN-γ, IL-1ß and TNF-α highly increased CXCL11 mRNA expression and CXCL11 protein secretion by ARPE-19 cells. Resveratrol substantially inhibited the proinflammatory cytokines-induced CXCL11 production while partially blocking nuclear factor-κB activation. This inhibitory action of resveratrol was also observed for the cytokines-induced expression of chemokines CXCL9, CCL2 and CCL5. Our results indicate that resveratrol could potentially attenuate RPE inflammatory response implicated in the pathogenesis of AMD.

Resveratrol Suppresses Expression of VEGF by Human Retinal Pigment Epithelial Cells: Potential Nutraceutical for Age-related Macular Degeneration.

Age-related macular degeneration (AMD) is a sight threating retinal eye disease that affects millions of aging individuals world-wide. Choroid-retinal pigment epithelium (RPE)-neuroretina axis in the posterior compartment of the eye is the primary site of AMD pathology. There are compelling evidence to indicate association of vascular endothelial growth factors (VEGF) to AMD. Here, we report the inhibitory actions of resveratrol (RSV) on inflammatory cytokine, TGF-ß and hypoxia induced VEGF secretion by human retinal pigment epithelial cells (HRPE). HRPE cultures prepared from aged human donor eyes were used for the studies in this report. HRPE secreted both VEGF-A and VEGF-C in small quantities constitutively. Stimulation with a mixture of inflammatory cytokines (IFN-γ, TNF-α, IL-1ß), significantly increased the secretion of both VEGF-A and VEGF-C. RSV, in a dose dependent (10-50 uM) manner, suppressed VEGF-A and VEGF-C secretion induced by inflammatory cytokines significantly. RT-PCR analysis indicated that effects of RSV on VEGF secretion were possibly due to decreased mRNA levels. TGF-ß and cobalt chloride (hypoxia mimic) also upregulated HRPE cell production of VEGF-A, and this was inhibited by RSV. In contrast, RSV had no effect on anti-angiogenic molecules, endostatin and pigment epithelial derived factor secretion. Studies using an in vitro scratch assay revealed that wound closure was also inhibited by RSV. These results demonstrate that RSV can suppress VEGF secretion induced by inflammatory cytokines, TGF-ß and hypoxia. Under pathological conditions, over expression of VEGF is known to worsen AMD. Therefore, RSV may be useful as nutraceutical in controlling pathological choroidal neovascularization processes in AMD.