Santosh K. Bharti

Detection of Pancreatic Cancer–Induced Cachexia Using a Fluorescent Myoblast Reporter System and Analysis of Metabolite Abundance

The dire effects of cancer-induced cachexia undermine treatment and contribute to decreased survival rates. Therapeutic options for this syndrome are limited, and therefore efforts to identify signs of precachexia in cancer patients are necessary for early intervention. The applications of molecular and functional imaging that would enable a whole-body holistic approach to this problem may lead to new insights and advances for diagnosis and treatment of this syndrome. Here we have developed a myoblast optical reporter system with the purpose of identifying early cachectic events. We generated a myoblast cell line expressing a dual tdTomato:GFP construct that was grafted onto the muscle of mice-bearing human pancreatic cancer xenografts to provide noninvasive live imaging of events associated with cancer-induced cachexia (i.e., weight loss). Real-time optical imaging detected a strong tdTomato fluorescent signal from skeletal muscle grafts in mice with weight losses of only 1.2% to 2.7% and tumor burdens of only approximately 79 to 170 mm(3). Weight loss in cachectic animals was also associated with a depletion of lipid, cholesterol, valine, and alanine levels, which may provide informative biomarkers of cachexia. Taken together, our findings demonstrate the utility of a reporter system that is capable of tracking tumor-induced weight loss, an early marker of cachexia. Future studies incorporating resected tissue from human pancreatic ductal adenocarcinoma into a reporter-carrying mouse may be able to provide a risk assessment of cachexia, with possible implications for therapeutic development.

Metabolic consequences of HIF silencing in a triple negative human breast cancer xenograft

Hypoxia is frequently encountered in tumors and results in the stabilization of hypoxia inducible factors (HIFs). These factors transcriptionally activate genes that allow cells to adapt to hypoxia. In cancers, hypoxia and HIFs have been associated with increased invasion, metastasis, and resistance to chemo and radiation therapy. Here we have characterized the metabolic consequences of silencing HIF-1α and HIF-2α singly or combined in MDA-MB-231 triple negative human breast cancer xenografts, using non-invasive proton magnetic resonance spectroscopic imaging (1H MRSI) of in vivo tumors, and high-resolution 1H MRS of tumor extracts. Tumors from all three sublines showed a significant reduction of growth rate. We identified new metabolic targets of HIF, and demonstrated the divergent consequences of silencing HIF-1α and HIF-2α individually on some of these targets. These data expand our understanding of the metabolic pathways regulated by HIFs that may provide new insights into the adaptive metabolic response of cancer cells to hypoxia. Such insights may lead to novel metabolism based therapeutic targets for triple negative breast cancer.

Hypoxia-induced reporter genes with different half-lives

The utility of reporter genes has gained significant momentum over the last three decades. Reporter genes are used to understand the transcriptional activity of a gene both in vitro and in vivo, and in pathway analysis and drug screening for diseases involving protozoan parasites, and in anti-cancer drug developments. Here, using a human prostate cancer xenograft model (PC3), we describe a method to construct and validate hypoxia reporter genes with different half-lives. Using molecular biology and optical imaging techniques, we have validated the expression of long half-life enhanced green fluorescence protein (EGFP) expression and short half-life luciferase gene expression to report on the spatial and temporal evolution of hypoxia in vivo.

Metabolomic characterization of experimental ovarian cancer ascitic fluid

IntroductionMalignant ascites (MA) is a major cause of morbidity that occurs in 37% of ovarian cancer patients. The accumulation of MA in the peritoneal cavity due to cancer results in debilitating symptoms and extremely poor quality of life. There is an urgent unmet need to expand the understanding of MA to design effective treatment strategies, and to improve MA diagnosis.ObjectiveOur purpose here is to contribute to a better characterization of MA metabolic composition in ovarian cancer.MethodWe determined the metabolic composition of ascitic fluids resulting from orthotopic growth of two ovarian cancer cell lines, the mouse ID8- vascular endothelial growth factor (VEGF)-Defb29 cell line and the human OVCAR3 cell line using high-resolution 1H MRS. ID8-VEGF-Defb29 tumors induce large volumes of ascites, while OVCAR3 tumors induce ascites less frequently and at smaller volumes. To better understand the factors driving the metabolic composition of the fluid, we characterized the metabolism of these ovarian cancer cells in culture by analyzing cell lysates and conditioned culture media with 1H NMR.ResultsDistinct metabolite patterns were detected in ascitic fluid collected from OVCAR3 and ID8-VEGF-Defb29 tumor bearing mice that were not reflected in the corresponding cell culture or conditioned medium.ConclusionHigh-resolution 1H NMR metabolic markers of MA can be used to improve characterization and diagnosis of MA. Metabolic characterization of MA can provide new insights into how MA fluid supports cancer cell growth and resistance to treatment, and has the potential to identify metabolic targeting strategies to reduce or eliminate the formation of MA.

Abstract 2509: High-resolution1H human plasma profiling of pancreatic ductal adenocarcinoma

Early detection of pancreatic ductal carcinoma (PDAC) is critically important because by the time PDAC is detected almost 80% of patients are surgically unresectable1. While imaging with CT, MRI and ultrasound is making significant inroads in PDAC detection, the costs associated with imaging impose a barrier for routine screening. Plasma based detection of PDAC would provide a relatively inexpensive and easy method for routine screening for the purpose of early detection. Metabolic profiling of plasma samples using high resolution 1H MRS provides an opportunity to assist in the detection of PDAC. Here we characterized the plasma metabolome of normal subjects, subjects with benign pancreatic disease, and subjects with PDAC to evaluate the ability of 1H MRS to identify metabolic changes in plasma associated with PDAC. Plasma from patients with PDAC (n=4), patients with benign pancreatic disease (n=2) and from healthy control subjects (n=4) were included in this study. Final diagnosis was established by histopathological evaluation of surgical specimens. Three hundred micro liter of D2O phosphate buffer saline (NaCl 0.9% in 90% D2O) was mixed with 300 μL of plasma and transferred to 5 mm NMR tubes. High-resolution 1H MRS was performed on an Avance 750 MHz Bruker MR spectrometer. The Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence with water suppression [PRESET-90°-(d-180-d)n-Aq] was performed to remove short T2 components arising due to the presence of macromolecules2. The metabolites were identified and quantified from 1H MRS spectra from the three groups. Even with a small sample size clear differences were identified in metabolites such as lactate, pyruvate, β-hydroxybutyrate, acetate and acetoacetate in plasma from PDAC subjects compared to normal subjects. The increase of lactate and pyruvate detected in plasma may reflect altered glucose metabolism occurring in PDAC. β-hydroxybutyrate, acetate and acetoacetate are involved in ketone body synthesis and may reflect altered ketone body metabolism occurring in PDAC. In a recent study, higher levels of ketone bodies and lactate were detected in the serum of rats with 7,12-dimethylben(a)anthracen (DMBA)-induced pancreatic intraepithelial neoplasia (PanIN) compared to plasma. Interestingly, with progression to PDAC several of these metabolites decreased3. Our data support further investigation of 1H MRS of human plasma to detect PDAC in combination with techniques such as circulating tumor cell phenotyping References: (1) Kotteas, E., et al. J Cancer Res Clin Oncol 2016, 142, 1795-1805. (2) Van, Q. N., et al. Biochem Biophys Res Commun 2003, 301, 952-959. (3) Lin, X., et al. Mol Biosyst 2016, 12, 2883-2892. This work was supported by NIH R01 CA193365. Citation Format: Santosh K. Bharti, Michael Goggins, Zaver M. Bhujwalla. High-resolution 1H human plasma profiling of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2509. doi:10.1158/1538-7445.AM2017-2509

Abstract 2505: High-resolution MRS characterization of malignant ascites in two models of ovarian cancer

Build-up of malignant ascites occurs in more than one third of ovarian cancer patients and significantly contributes to poor quality of life and mortality. Advances in understanding malignant ascites formation and finding new therapeutic options are urgently needed. High-resolution 1H MRS has been described as extremely useful in detecting endogenous metabolites to diagnose cancer, by providing a detailed overview of metabolic pathways in a single measurement. In the present study, we are using two ovarian cancer cell lines, the murine ID8-VEGF-Defb29 cell line and the human OVCAR3 cell line. Implanted orthotopically, these two ovarian cancer models are characterized by different profile of ascites formation. The mouse cell line ID8-VEGF-Defb29 induces large volumes of ascites, often more than 10 mL, while the human OVCAR3 cell line induces ascites less frequently and at smaller volumes, usually less than 0.2 mL. We applied high-resolution 1H MRS to compare the metabolic composition of both ascitic fluids. To better understand the differences observed, we characterized the metabolism of these ovarian cancer cells in culture by analyzing cell lysates and conditioned culture media with 1H MRS to advance our understanding of cancer cell metabolic reprogramming in malignant ascites formation and the role of the tumor microenvironment in ascites formation and composition. The two tumor models used in this study induced different ascitic profiles. While OVCAR3 tumor bearing mice developed small viscous volume of ascites, ID8-VEGF-Defb29 induced higher volumes. ID8-VEGF-Defb29 ascitic fluids were characterized by higher levels of glutamine, glucose, poly-unsaturated fatty acids and pyruvate compared to the OVCAR3 fluids, while all the other metabolites, including glutamate, lactate, myo-inositol, choline and acetate, were lower. To determine if the differences observed in the ascitic fluids were only due to a different metabolism of the cancer cells, we investigated their metabolism in vitro. We analyzed the metabolites present in the conditioned cell culture media, and in the cells, and observed differences in OVCAR3 and ID8-VEGF-Defb29 cells metabolism in vitro, without replicating the differences observed in vitro. A Venn diagram of the different metabolites present in the cells, media and ascites showed differences in the metabolites present in those 3 compartments, and highlighted the ones in common. Beta-hydroxybutyrate, lipids, maleic and citrate were found in both ascites, and not in the cells or media. Ascites MRS derived biomarkers could help in ovarian cancer diagnosis, and enhance our understanding of the biochemical and metabolic changes associated with ovarian cancer, and with ascites formation. Supported by Tina’s Wish Foundation, NIH P50CA013175 and P30CA06973. Citation Format: Santosh Bharti, Flonne Wildes, Chien-Fu Hung, TC Wu, Zaver Bhujwalla, Marie-France Penet. High-resolution MRS characterization of malignant ascites in two models of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2505. doi:10.1158/1538-7445.AM2017-2505

Abstract 1185: Altered choline metabolism in pancreatic cancer cells and tumor xenografts

Early-stage pancreatic cancer is usually clinically silent, and most patients presenting symptoms attributable to pancreatic cancer have advanced disease. Better understanding of the disease, effective detection methods, and new therapeutic targets are urgently needed to improve its outcome. Magnetic resonance spectroscopy (MRS) is being evaluated in the diagnosis of several solid malignancies (1). A hallmark of most solid tumors is the detection of elevated levels of phosphocholine (PC) and total choline (tCho) (1). The increase in PC levels in solid tumors has been attributed to high level of choline kinase (Chk)-alpha (2). Here, we characterized the metabolism of multiple pancreatic ductal adenocarcinoma cell lines and tumors with in vitro high resolution MRS and in vivo 1H magnetic resonance spectroscopic imaging (MRSI). We also characterized the expression of Chk-alpha in the cells and tumor extracts. Eight pancreatic adenocarcinoma cell lines (Panc1, BxPC3, Pa04C, Pa02C, Pa20C, Pa28C, Pa03C, and Pa09C) and one immortalized pancreatic cell line (human pancreatic nestin expressing (HPNE)) were investigated. For the in vivo experiments, cells were inoculated subcutaneously in male SCID mice. Once tumors reached 500 mm3, the mice were scanned on a 4.7T spectrometer for 1H MRSI, after which the tumors excised for immunoblot analysis, and high-resolution 1H MRS. Cells and tumor extracts were carried out as previously described (3). Fully relaxed 1H MR spectra of the extracts were acquired on a Bruker Avance 500 spectrometer at 11.7 T. Integrals of the metabolites of interest were determined and normalized to the number of cells and to the tumor weight respectively. Significant differences in tCho were identified in pancreatic cell lines and the tumor xenografts. In the panel of cell lines, the differences in tCho levels were associated with differences in Chk-alpha expression. PC and tCho levels were significantly elevated in the pancreatic cancer cell lines compared to the immortalized pancreatic cell line, and Chk-alpha was overexpressed in the cancer cell lines. In vivo studies revealed that elevated tCho was detected in tumors derived from the 4 cell lines tested, with higher levels of tCho in Panc1 tumors. These in vivo results were confirmed by ex vivo MRS analysis of the tumor extracts. The high level of tCho observed in Panc1 tumors was mainly due to increased PC, and correlated with high Chk-alpha expression observed in the immunoblots. These data support the use of 1H MRS to noninvasively detect pancreatic cancer. Moreover, the aberrant choline metabolism may provide novel targets in the treatment of pancreatic cancer. Acknowledgement: This work was supported by NIH P50CA103175. *Equal contribution. References: (1) Glunde et al., Nature reviews (2006). (2) Ramirez de Molina et al., Biochem Biophys Res Commun (2002). (3) Shah et al., NMR Biomed (2012). Citation Format: Marie-France Penet*, Tariq Shah*, Santosh Bharti, Yelena Mironchik, Flonne Wildes, Anirban Maitra, Zaver M. Bhujwalla. Altered choline metabolism in pancreatic cancer cells and tumor xenografts. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1185. doi:10.1158/1538-7445.AM2014-1185