Rattan, Ramandeep

Abstract Introduction Ovarian cancer (OvCa) remains the most lethal gynecologic malignancy, with older women experiencing the poorest outcomes. Aging profoundly remodels the ovarian tumor immune microenvironment (TME), promoting regulatory T cell (Treg) mediated immune evasion; however, the underlying metabolic mechanisms remain unclear. Methods Using young (3-month) and aged (22-month) female mice injected intraperitoneally with ID8p53⁻/⁻ OvCa cells, we investigated how age associated metabolic reprogramming influences tumor immunity Results Aged OvCa bearing mice exhibited accelerated tumor progression, reduced survival (median 54 vs. 72 days), decreased CD4+ and CD8+ T cells, and an expanded Treg population. Metabolomic profiling revealed increased succinate accumulation in ascites, tumor tissue, and serum of aged mice and elderly OvCa patients. Exogenous succinate enhanced CCR4+FOXP3+ Treg frequency and upregulated IL-10 and TGF-β secretion. Elevated levels of CCL22, a CCR4 ligand, were also detected in ascites from aged mice and patients, suggesting a chemokine-driven reinforcement of Treg recruitment. PBMCs from aged patients displayed an OXPHOS-reprogrammed CCR4+FOXP3+ Treg subset with heightened suppressive capacity, markedly inhibiting CD4+ and CD8+ effector cytotoxicity. Treatment with AA6, an α-ketoglutarate dehydrogenase inhibitor that blocks succinate synthesis, reduced CCR4+FOXP3+ Treg accumulation, restored effector T-cell function, and significantly prolonged survival in aged OvCa-bearing mice. Conclusion Together, these findings reveal a novel succinate—CCR4 metabolic—chemokine axis that drives Treg mediated immunosuppression in aged OvCa. Targeting this pathway offers a promising strategy to restore anti-tumor immunity and overcome age-related immunotherapy resistance in ovarian cancer. Funding Source N/A Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

ABSTRACT Epithelial ovarian cancer (EOC) incidence and mortality increase with age, driven in part by chronic inflammation, diminished T cell output, and heightened regulatory T cell (Treg) mediated immunosuppression. In aged EOC‐bearing mice, we observed reduced survival, accompanied by impaired CD4 + and CD8 + T cell responses and a marked expansion of FOXP3 + Tregs exhibiting elevated IL‐10 and TGFβ expression. Metabolic profiling revealed enhanced oxidative phosphorylation in Tregs from aged mice, along with a fivefold increase in intracellular succinate levels. This accumulation of succinate within the aged tumor microenvironment was found to potentiate Treg suppressive function. Notably, pharmacologic inhibition of α‐ketoglutarate dehydrogenase reversed this effect, restoring effector T cell activity. These findings highlight succinate‐driven metabolic reprogramming as a central mechanism of age‐related Treg dysfunction in EOC and suggest that targeting succinate metabolism may offer a promising strategy to rejuvenate antitumor immunity in elderly patients.

Abstract Multiple sclerosis is a chronic inflammatory and demyelinating disease of the CNS. Although, MS is well characterized in terms of the role played by immune cells, cytokines and CNS pathology, nothing is known about the metabolic alterations that occur during the disease process in circulation. Here, we profiled the plasma metabolites of B6 with EAE, a known model of chronic MS using untargeted global metabolomics. Using a combination of high-throughput liquid-and-gas chromatography with mass spectrometry, a total of 326 metabolites were identified, with significant changes observed in 105 metabolites (13 up- and 92 down-regulated), that mapped to lipid and amino acid pathways, followed by the peptide, xenobiotic, carbohydrate, nucleotide and energy pathways (p<0.05). To understand the functional role of these alterations, the KEGG metabolic library was analyzed using Metaboanalyst. The top nine most significant pathways in terms of GlobeTest p-value and impact are 1) fatty acid biosynthesis, 2) lysine degradation, 3) histidine metabolism, 4) beta-alanine metabolism, 5) pentose phosphate pathway, 6) arachidonic acid metabolism, 7) linoleic acid, 8) D-arginine and ornithine. Overall, these metabolic changes could be exploited as biomarkers for EAE/MS disease and to design new treatment paradigms where metabolic interventions could be combined with present and experimental therapeutics to achieve better treatment.

Abstract In search of metabolites signature as biomarkers during EAE disease, we profiled urine from B6 EAE using global untargeted metabolomics. Evaluation of metabolomic profiling of urine from EAE and healthy B6 group by using a combination of high-throughput liquid-and-gas chromatography with mass spectrometry, we found that 132 out of 322 (41%) metabolites were differentially altered (P<0.05) indicating robust alteration in the urine metabolomics profile during disease. Among the perturbed metabolites, 11 were up regulated in EAE urine whereas 121 were down regulated. We conducted pathway analysis of the biochemical pathways of the KEGG and considered both concerted changes in metabolite intensity within the pathway (GlobalTest) and alterations of high impact, and found that a number of pathways were significantly altered including glyoxylate and dicarboxylate, phenylanine metabolism, porphyrin & chlorophyll metabolism, primary bile acid biosynthesis, cysteine & methione metabolism, taurine and hypotaurine metabolism, glycine, serine & threonine metabolism, and beta-alanine metabolism. Alteration in these pathways during EAE disease suggesting that perturbation of certain central metabolites could have impact on multiple metabolic pathways. While some of these metabolite changes could easily be developed as biomarkers, the key to translating metabolomics into therapeutics would require figuring out the central altered metabolic pathway(s), once studied in detail.

Abstract AMPK is an energy-sensing metabolic switch implicating in various metabolic disorders. Here we report its new role in controlling the immunopathology of experimental autoimmune encephalitis by polarizing pro-inflammatory phenotype of Macrophage. AMPK alpha1 knockout (alpha1KO) developed severe EAE due to higher expression of IL17 with reduced IFNy and IL4. AMPK alpha1KO also exhibited increased number of infiltrated mononuclear cells in CNS due to impaired blood brain barrier integrity. Macrophage from alpha1KO mice exhibited M1 type phenotype (IL10lowIL12/23high) with stronger capacity to induce allogenic and antigen-specific T-cell response and also heightened the encephalitogenic property of MOG35-55-primed CD4 T cells in B6 mice. Collectively, our data shows AMPK controls the clinical outcome of disease by regulating M1 phenotype-Th17 axis in animal model of MS.

Abstract AMPK is an energy sensing metabolic switch in mammalian cells and regulates diverse metabolic pathway in cells. Recently, we observed that AMPK is lost in all immune cells of Experimental autoimmune encephalomyelitis (EAE), widely used animal model for MS. AMPK is lost at protein level in spleen macrophages, total T cells and their subsets (CD4, CD8 and regulatory T cells) isolated from EAE afflicted animals compared to control. To examine the role of AMPK, EAE was induced in Wt (+/+) and AMPKalpha1 null mice (-/-) and found that AMPKalpha1 (-/-) mice exhibited severe EAE disease. AMPKalpha2 is not involved in enhancing the severity of the disease. Moreover, AMPKalpha1 null mice significantly exhibited severe inflammation and demyelination in CNS. Loss of AMPKalpha1 leads to higher production of IL17 cytokine and reduced production of IFNgamma and IL4 under recall response with MOG35-55 as antigen. AMPKalpha1 KO mice exhibited higher number of infiltrated mononuclear cells in CNS and produced higher amount of IL17 significantly compared to Wt mice. Moreover, adoptive transfer of MOG35-55 specific T cells from AMPKalpha1 KO in Wt mice (KO to Wt) induced significantly higher degree of clinical disease compared to adoptive transfer of MOG specific T cells from Wt to Wt mice suggesting loss of AMPK negatively regulate clinical disease of EAE and identify AMPK as a novel therapeutic target for MS.

Abstract The AMP-activated protein kinase, AMPK, is an energy-sensing, metabolic switch implicated in various metabolic disorders; however, its role in inflammation is not well defined. We have previously shown that loss of AMPK exacerbates experimental autoimmune encephalomyelitis (EAE) disease severity. In this study, we investigated the mechanism through which AMPK modulates inflammatory disease like EAE. AMPKα1 knockout (α1KO) mice with EAE showed severe demyelination and inflammation in the brain and spinal cord compared with wild-type due to higher expression of proinflammatory Th17 cytokines, including IL-17, IL-23, and IL-1β, impaired blood–brain barrier integrity, and increased infiltration of inflammatory cells in the CNS. Infiltrated CD4 cells in the brains and spinal cords of α1KO with EAE were significantly higher compared with wild-type EAE and were characterized as IL-17 (IL-17 and GM-CSF double-positive) CD4 cells. Increased inflammatory response in α1KO mice was due to polarization of macrophages (Mϕ) to proinflammatory M1 type phenotype (IL-10lowIL-23/IL-1β/IL-6high), and these M1 Mϕ showed stronger capacity to induce allogenic as well as Ag-specific (myelin oligodendrocyte glycoprotein [MOG]35–55) T cell response. Mϕ from α1KO mice also enhanced the encephalitogenic property of MOG35–55–primed CD4 T cells in B6 mice. The increased encephalitogenic MOG-restricted CD4+ T cells were due to an autocrine effect of IL-1β/IL-23–mediated induction of IL-6 production in α1KO Mϕ, which in turn induce IL-17 and GM-CSF production in CD4 cells. Collectively, our data indicate that AMPK controls the inflammatory disease by regulating the M1 phenotype–Th17 axis in an animal model of multiple sclerosis.

Abstract A substantial body of evidence suggests an association between vascular endothelial growth factor (VEGF) and the neovascularization that occurs with ovarian carcinogenesis. VEGF expression has been directly related to ascites accumulation. Phase II clinical trials in ovarian cancer using single-agent bevacizumab (a monoclonal antibody directed against VEGF) showed promising results, clearly highlighting the importance of anti-angiogenesis based-targeted therapies in ovarian cancer. One of the ways that VEGF mediates angiogenesis is by activating NADPH oxidase-ROS pathway. We have developed and engineered nanoparticles (NP) of cerium oxide (Nanoceria, NCe), a rare earth oxides, which have been tested for their ability to serve as free-radical scavengers to provide protection against chemical, biological, and radiological insults that promote the production of free radicals. Here we show for the first time that treatment of human umbilical vascular endothelial cells (HUVECs) with NCe resulted in inhibition of proliferation, capillary tube formation and VEGF165 induced activation of VEGFR2 and MMP2. Additionally, NCe treatment inhibited growth factor induced migration and invasion of ovarian cancer cells. Collectively, these results indicate that cerium oxide based NP could potentially be used as an antiangiogenic therapeutic agent in ovarian cancer. Citation Information: Cancer Res 2009;69(23 Suppl):C48.

Abstract Metformin is the most widely used drug for type 2 diabetes. Metformin activates AMP-activated protein kinase (AMPK), which regulates cellular energy metabolism via its upstream kinase LKB1. Activation of AMPK has been shown extensively to cause growth arrest in multiple tumor types by inhibiting the TSC-mTOR (mammalian target of rapamycin) pathway as well as by inducing cell cycle arrest. Our data indicate that metformin exerts an anti-proliferative effect on several chemo-responsive and -resistant ovarian cancer cell lines (A2780, CP70, C200, OV202, OVCAR3 and SKVO3ip, PE01, PE04), which is accompanied by activation of AMPK and cell cycle arrest. Interestingly, we have also determined that metformin is able to inhibit proliferation of ovarian cancer cells when AMPK is silenced by siRNA, where both the expression and activation of AMPK is significantly reduced. Also, metformin was able to attenuate proliferation of AMPK (−/−) mefs, although to a lesser degree in AMPK (+/+) mefs. Additional results indicate that metformin activates p21 and inhibits cyclinD1 activities partially in AMPK (−/) mefs. These data suggest a novel mechanism of metformin action, namely, an AMPK independent pathway which can modulate proliferation and possibly cell cycle regulated events in ovarian cancer. Whether the AMPK independent effects of metformin require LKB1 activity is currently under investigation. Collectively, our preliminary observations indicate that metformin can modulate proliferation and cell cycle arrest in an AMPK independent manner. Citation Information: Cancer Res 2009;69(23 Suppl):C38.

Abstract Objectives. To assess chemo-response of patient derived ovarian cancer cells to first line chemotherapeutic agents as well as targeted therapies. Methods. Ascites was obtained at laparotomy from 14 patients with epithelial ovarian cancer. Short term cultures were established in 11 of these and the cells characterized by morphology and immuno-staining to cytokeratin AE1/AE3 and E-cadherin. MTT assays were done on the 11 samples after 48 hours of exposure to Cisplatin, Paclitaxel, Vandetanib and Flavopiridol. Results. Three samples did not grow well and ultimately showed senescence or were over-run by fibroblasts. In the remaining 11 samples, morphology showed the cells to be arranged in a cobble-stone or rosette pattern. Most of the cells stained positive for cytokeratin AE1/AE3 and E-cadherin. The majority of samples (8/11, 73%) were sensitive to either Cisplatin or Paclitaxel. Of the three resistant samples, one was sensitive to Vandetanib, one to Flavopiridol and one was resistant to all four drugs. Two patients did not receive chemotherapy due to morbidity or mortality. Among those who were treated, 4 (50%) had complete response and are currently free of disease. Conclusions. Short term cultures may be a good method of studying chemo-response of tumor cells ex-vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3544.