Santosh K. Bharti

Cancer-induced cachexia accounts for approximately 20% of all cancer deaths [1] and affects nearly 80% of pancreatic cancer patients [2, 3]. Cachectic patients experience a wide range of symptoms affecting several organ functions such as muscle, liver, brain, and heart that decrease quality of life and worsen prognosis. A major characteristic of cachexia is the accelerated skeletal muscle and fat storage wasting causing nutrient mobilization both directly as lipid and amino acids, and indirectly as glucose derived from the exploitation of liver gluconeogenesis that reaches the tumor through the bloodstream [4]. Here, for the first time, we have performed high-resolution quantitative 1H magnetic resonance spectroscopy (MRS) of spleen tissue obtained from normal mice and mice bearing PDAC that are cachectic (Pa04C) and non-cachectic (Panc1). The Panc1 (non-cachectic), and Pa04C (cachectic) PDAC cell lines were used [5]. Eight-week-old male severe combined immunodeficient mice were inoculated in the right flank with cancer cells (5×106). Mice were euthanized once tumors were ~ 300 mm3. Control, cachectic and non-cachectic groups consisted of 9, 10 and 9 mice per group respectively. Spleen water soluble metabolites were extracted, freeze dried, reconstituted in D2O PBS and transferred to an NMR tube for spectral acquisition with a 750 MHz (17.6T) MR spectrometer [6]. As anticipated, mice with cachexia-inducing Pa04C tumors showed significant weight loss with time. We observed, for the first time, that spleens from cachectic Pa04C tumor bearing mice showed a profound weight loss compared to normal mice and mice with Panc1 tumors. However, an increase of liver and spleen size has been previously reported in terminal cachectic human patients from colorectal cancer measured by CT scan 2-11 months prior to death [7]. A significant decrease in almost all amino acids was observed in cachectic (Pa04C) mouse spleens compared to normal and non-cachectic (Panc1) mouse spleens. Differences in choline metabolites, creatine, glutamine, glutamate, glutathione and aspartate were observed in cachectic mouse spleens compared to non-cachectic mouse spleens and spleens from healthy control mice. The significant decrease of amino acids in the cachectic spleens may reflect increased utilization of amino acids by the tumor or other organs during the cachexia muscle/protein wasting [4]. These results provide new insights into changes in spleen metabolism during cachexia, and support investigating metabolic targets to reduce cachexia associated morbidity. Supported by NIH R01CA193365 and R35CA209960. Reference: 1. Argiles et al. Nat. Rev. Cancer 2014, 14 (11), 754; 2. Fearon et al. HPB (Oxford) 2010, 12 (5), 323; 3. Ozola et al. Pancreatology 2015, 15 (1), 19; 4. Porporato et al. Oncogenesis 2016, 5, e200; 5. Winnard et al. Can. Res. 2016, 76(6): 1441; 6. Penet et al. Clin. Can. Res. 2015, 21 (2), 386; 7. Lieffers et al. Am J Clin Nutr. 2009, 89 (4), 1173 Citation Format: Santosh Kumar Bharti, Paul T. Winnard, Raj Kumar Sharma, Yelena Mironchik, Marie-France Penet, Zaver M. Bhujwalla. Spleen metabolism altered by human pancreatic cancer xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5265.

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...

Supplemental Figure S1. Real time optical imaging after 4 weeks of To3B graft growth in the hind leg muscle of a nude mouse indicating that a tdT fluorescent signal was observed only after i.p. administration of 100 μg of DEX 24 hr prior to imaging the live animal.

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.

Introduction: Cancer-induced cachexia has lethal consequences. Cachexia results in lower tolerance to chemotherapy1. Cachectic patients experience a wide range of symptoms affecting the function of organs such as muscle, liver, brain, and heart, causing significant morbidity 2. An altered choline metabolism is one of the hallmarks of cancers. Here, for the first time, we identified changes in brain choline metabolism induced by cachexia-inducing pancreatic cancer xenografts and related these changes to plasma choline levels. Methods: Panc1 or Pa04C cells were implanted in the flank of SCID mice. Snap frozen brains were powdered under liquid nitrogen, weighed, and dual phase extraction was performed3. All 1H MR spectra were acquired on a Bruker Avance III 750 MHz MR spectrometer. Mouse plasma choline was quantified using a fluorometric assay (Choline Assay Kit, BioAssay Systems). Brain and plasma studies were performed with normal mice (n=10), mice with cachexia-inducing Pa04C tumors...

Introduction: Cachexia is a complex metabolic syndrome that affects nearly 80% of pancreatic ductal adenocarcinoma (PDAC) patients. Major symptoms of cancer induced cachexia include loss of skeletal muscle weight with or without fat loss and reduced physical activity [1, 2]. Cancer cachexia may lead to multiple organ dysfunction and metabolic abnormalities [3]. To date there are no known cures for this condition. Our ongoing studies have focused on understanding the metabolic impact of PDAC induced cachexia in vital organs using 1H magnetic resonance spectroscopy (MRS) of organ extracts to expand our understanding of this syndrome. Here we have focused on understanding the metabolic changes occurring in the lungs and kidneys using our well established PDAC xenograft models. We compared organ changes in mice with cachexia-inducing xenografts (Pa04C) to mice with non-cachexia-inducing xenografts (Panc1) and normal mice. We have previously established significant weight loss in Pa04C t...

Adipocytic tumors present a spectrum of neoplastic disease including benign lipomas, atypical lipomatous tumors (ALTs), and malignant liposarcomas. Liposarcomas are the most common soft tissue sarcoma, accounting for approximately 20% of all adult soft tissue sarcomas [1]. Some liposarcomas are suspected to arise through dedifferentiation of ALTs, a process that is not well understood. Pleomorphic liposarcomas are high-grade, aggressive tumors with high metastatic potential and overall 5-year survival rate of 20-30% [3]. Distinguishing high-grade liposarcomas from benign and atypical lipomatous tumors can be a diagnostic challenge with implications for surgical and clinical management. The rate of misdiagnosis is approximately 30–40% following radiological detection [4], and 7–17% on histological evaluation [5]. De-identified human surgical samples were collected from the Dept. of Orthopaedic Surgery, JHU, School of Medicine Baltimore, MD. One tissue specimen from a benign pleomorph...

Cancer-induced cachexia accounts for approximately 20% of all cancer deaths 1. In pancreatic cancer, the syndrome affects nearly 80% of patients 2,3. Cachectic patients experience a wide range of symptoms affecting the function of several organs such as muscle, liver, brain, and heart, that decrease quality of life and worsen prognosis. A major characteristic of cachexia is the accelerated skeletal muscle and fat storage wasting causing nutrient mobilization both directly as lipid and amino acids, and indirectly as glucose derived from the exploitation of liver gluconeogenesis that reaches the tumor through the bloodstream4. Patients with cachexia develop a wide range of metabolic stress from increased proteins and fat tissue burning resulting in increased energy expenditure. Here, for the first time, we have performed high-resolution quantitative 1H magnetic resonance spectroscopy (MRS) of liver tissue obtained from normal, noncachectic and cachectic mice bearing PDAC that are cach...

Cachexia is an underexplored and yet devastating consequence of cancer that is the cause of 20% of all cancer related deaths1. Cachexia inducing tumors cause a ‘wasting away’ of the body. The condition is associated with poor treatment outcome2, fatigue, and extremely poor quality of life2,3. Because of the multi-factorial characteristics of this condition, it has been difficult to understand the mechanisms driving the impact of the tumor on body organs and the sequence of events that leads to this lethal condition. Here we have used 1H MRS to characterize the metabolic profile of tumor interstitial fluid (TIF) obtained from noncachexia (Panc1) and cachexia inducing (Pa04C) tumors to further understand the impact of the deranged metabolism of cachexia-inducing tumors on the tumor metabolic secretome. The human pancreatic cancer cell line, Panc1, was obtained from ATCC. The human pancreatic cancer cell line, Pa04C, was provided by Dr. Maitra4. Six to 8 week old male SCID mice were in...

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 ...

One of the least examined, and yet critically important factors of the tumor microenvironment (TME) is the tumor interstitial fluid (TIF) that contains the tumor secretome. This fluid surrounds cancer and stromal cells and contains various cytokines, and nutritional and molecular factors that shape the outcome of nearly all aspects of tumor angiogenesis, growth, metastasis, and response to treatment. As mining of targets to treat cancer expands into the TME, the TIF represents an important source of identifying new targets in cancer treatment. Angiogenesis, one of the major hallmarks of cancer, is essential for cancers to establish vasculature for growth and hematogenous metastasis. To further understand the TIF and plasma metabolite content in breast cancer, and the role of VEGF in modifying metabolite concentrations in the TME, here we used 1H MRS to characterize metabolites in TIF collected from the triple negative MDA-MB-231 and the ER-positive MCF-7 human breast cancer xenograf...

Cancer cells display an adaptive response to hypoxia through the activation of several genes mediated by the binding of hypoxia inducible factors (HIFs) to hypoxia response elements (HRE) in the promoter region of target gene that results in their increased transcription [1]. HIFs promote key steps in tumorigenesis, including angiogenesis, metabolism, proliferation, metastasis, and differentiation [1]. Bacterial or yeast cytosine deaminase (yCD) converts the nontoxic prodrug 5-fluorocytosine (5-FC) to the anti-cancer drug 5-fluorouracil (5-FU) that is widely used in cancer treatment [2]. Using a lentivirus approach, we established controlled expression of yCD by HRE in prostate cancer cells (PC-3). These cells also report on HIF-1α expression with regulated luciferase (Luc) expression, allowing detection of hypoxia, and the generation of 5-FU from 5-FC by yCD in the presence of hypoxia. Transduction efficiency and reporter activity in response to hypoxia was evaluated by performing ...

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 grafts in mice with weight loses of only 1.2 t...

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal disease that develops relatively symptom-free and is therefore advanced at the time of diagnosis. The absence of early symptoms and effective treatments has created a critical need for identifying and developing new noninvasive biomarkers and therapeutic targets. Experimental Design: We investigated the metabolism of a panel of PDAC cell lines in culture and noninvasively in vivo with 1H magnetic resonance spectroscopic imaging (MRSI) to identify noninvasive biomarkers and uncover potential metabolic targets. Results: We observed elevated choline-containing compounds in the PDAC cell lines and tumors. These elevated choline-containing compounds were easily detected by increased total choline (tCho) in vivo, in spectroscopic images obtained from tumors. Principal component analysis of the spectral data identified additional differences in metabolites between immortalized human pancreatic cells and neoplastic...

ABSTRACT This review illustrates the need to use nuclear magnetic resonance (NMR) spectroscopy for the quantitative analysis of small molecules in their crude forms and in mixtures.We provide the basic concepts of quantitative NMR (qNMR), a brief description of important acquisition and processing parameters responsible for obtaining high-quality, reproducible NMR spectra in order to maximize accuracy, and the latest referencing techniques used for quantitative analysis.We also describe methods that are used for quantitative analysis including calibration-curve and standard-addition methods. Further, we briefly address validation of qNMR spectroscopy and its major applications in various scientific disciplines.

The release of particulate pollutants into the air through burning of coal, crude oil, diesel, coal tar, etc. raises concerns of potential health hazards to the exposed human population. Polycyclic aromatic hydrocarbons (PAHs) are major toxic constituents of particulate matter (PM), which upon ingestion get metabolized to even more toxic metabolites such as quinones. The PAHs levels were assessed in both respirable particulate matter (RSPM, <10μM size) and suspended particulate matter (SPM, >10μM size) of urban ambient air (UAA) and that of major contributors viz. diesel exhaust particles (DEPs) and coal tar combustions emissions (CTCE). Seven US Environmental Protection Agency (USEPA) prioritized PAHs in RSPM and 10 in SPM were detected in UAA. Ten and 15 prioritized PAHs, respectively, were also detected in diesel exhaust particles (DEP) and coal tar combustion emission (CTCE) evidencing their release in the air. These PM associated PAHs for UAA, DEP and CTCE showed significant increase…

Glutamine addiction in c-MYC-overexpressing breast cancer is targeted by the aminotransferase inhibitor, aminooxyacetate (AOA). However the mechanism of ensuing cell death remains unresolved. A correlation between glutamine dependence for growth and c-MYC expression was studied in breast cancer cell lines. The cytotoxic effects of AOA, its correlation with high c-MYC expression, and effects on enzymes in the glutaminolytic pathway were investigated. AOA-induced cell death was assessed by measuring changes in metabolite levels by Magnetic Resonance Spectroscopy (MRS), the effects of amino acid depletion on nucleotide synthesis by cell cycle and BrdU uptake analysis, and activation of the endoplasmic reticulum (ER)-stress mediated pathway. Antitumor effects of AOA with or without common chemotherapies were determined in breast cancer xenografts in immunodeficient mice and in a transgenic MMTV-rTtA-TetO-myc mouse mammary tumor model. We established a direct correlation between c-MYC ov...

Purpose: Glutamine addiction in c-MYC–overexpressing breast cancer is targeted by the aminotransferase inhibitor, aminooxyacetate (AOA). However, the mechanism of ensuing cell death remains unresolved.

Experimental Design: A correlation between glutamine dependence for growth and c-MYC expression was studied in breast cancer cell lines. The cytotoxic effects of AOA, its correlation with high c-MYC expression, and effects on enzymes in the glutaminolytic pathway were investigated. AOA-induced cell death was assessed by measuring changes in metabolite levels by magnetic resonance spectroscopy (MRS), the effects of amino acid depletion on nucleotide synthesis by cell-cycle and bromodeoxyuridine (BrdUrd) uptake analysis, and activation of the endoplasmic reticulum (ER) stress–mediated pathway. Antitumor effects of AOA with or without common chemotherapies were determined in breast cancer xenografts in immunodeficient mice and in a transgenic MMTV-rTtA-TetO-myc mouse mammary tumor model.

Results: We established a direct correlation between c-MYC overexpression, suppression of glutaminolysis, and AOA sensitivity in most breast cancer cells. MRS, cell-cycle analysis, and BrdUrd uptake measurements indicated depletion of aspartic acid and alanine leading to cell-cycle arrest at S-phase by AOA. Activation of components of the ER stress–mediated pathway, initiated through GRP78, led to apoptotic cell death. AOA inhibited growth of SUM159, SUM149, and MCF-7 xenografts and c-myc–overexpressing transgenic mouse mammary tumors. In MDA-MB-231, AOA was effective only in combination with chemotherapy.

Conclusions: AOA mediates its cytotoxic effects largely through the stress response pathway. The preclinical data of AOA's effectiveness provide a strong rationale for further clinical development, particularly for c-MYC–overexpressing breast cancers. Clin Cancer Res; 21(14); 3263–73. ©2015 AACR.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal disease that develops relatively symptom-free and is therefore advanced at the time of diagnosis. The absence of early symptoms and effective treatments has created a critical need for identifying and developing new noninvasive biomarkers and therapeutic targets.

Experimental Design: We investigated the metabolism of a panel of PDAC cell lines in culture and noninvasively in vivo with 1H magnetic resonance spectroscopic imaging (MRSI) to identify noninvasive biomarkers and uncover potential metabolic targets.

Results: We observed elevated choline-containing compounds in the PDAC cell lines and tumors. These elevated choline-containing compounds were easily detected by increased total choline (tCho) in vivo, in spectroscopic images obtained from tumors. Principal component analysis of the spectral data identified additional differences in metabolites between immortalized human pancreatic cells and neoplastic PDAC cells. Molecular characterization revealed overexpression of choline kinase (Chk)-α, choline transporter 1 (CHT1), and choline transporter–like protein 1 (CTL1) in the PDAC cell lines and tumors.

Conclusions: Collectively, these data identify new metabolic characteristics of PDAC and reveal potential metabolic targets. Total choline detected with 1H MRSI may provide an intrinsic, imaging probe–independent biomarker to complement existing techniques in detecting PDAC. The expression of Chk-α, CHT1, and CTL1 may provide additional molecular markers in aspirated cytological samples. Clin Cancer Res; 21(2); 386–95. ©2014 AACR.

Curcumin has been reported to be therapeutically active but has poor bioavailability, half life, and high rate of metabolic detoxifcation. Most of the hydrophobic and acidic drugs get transported through human serum albumin (HSA). Binding of drugs to serum protein increases their half-life. The present study is focused to analyze interaction of curcumin with HSA by NMR and docking studies. In order to investigate the binding affinity of curcumin with HSA, NMR based diffusion techniques and docking study have been carried out. We report that curcumin has shown comparable binding affinity value vis-a-vis standard, the accessible surface area (ASA) of human serum albumin (uncomplexed) and its docked complex with curcumin at both binding sites was calculated and found to be close to that of warfarin and diazepam respectively. Conclusion drawn from our study demonstrates that curcumin interacts with HSA strongly thereby its poor half life is due to high rate of its metabolic detoxification as reported in literature. In diffusion NOE process the signals of small molecules remain (tryptophan and curcumin) which interact with macromolecules. However, the signals of molecules which do not interact disappear. This indicates curcumin and tryptophan molecule bind with human serum albumin

This review illustrates the need to use nuclear magnetic resonance (NMR) spectroscopy for the quantitative analysis of small molecules in their crude forms and in mixtures.

We provide the basic concepts of quantitative NMR (qNMR), a brief description of important acquisition and processing parameters responsible for obtaining high-quality, reproducible NMR spectra in order to maximize accuracy, and the latest referencing techniques used for quantitative analysis.

We also describe methods that are used for quantitative analysis including calibration-curve and standard-addition methods. Further, we briefly address validation of qNMR spectroscopy and its major applications in various scientific disciplines

A method of choosing the correction factor based on Bloch equation for the quantitative estimation of metabolite from 1H NMR spectra recorded with reduced recycle delay is prescribed. The procedure reduces the experimental time without substantially compromising the accuracy of quantitative estimation. It is based on choosing the correction factors, which depend on T1 and T2 of the metabolite and recycle delay used for recording the spectra. It is validated by studying a mixture of amino acids with known concentration of constituents and human serum sample and it provides accuracy of quantitative estimation to 95–96%.

Pus samples obtained from 109 patients with liver abscess were examined by NMR spectroscopy. To our knowledge this is the first report on metabolic profiling of liver abscesses. Fifty metabolites were identified by combination of one (1D) and two-dimensional (2D) NMR spectra. Metabolic derangements were evaluated for differentiation between amoebic (ALA) and pyogenic liver abscess (PLA). The NMR results indicate that aspartate, asparagine and galactose, integral components of lipoproteophophoglycans (LPG) of the cell wall of Entamoeba histolytica are metabolic biomarkers of ALA. On the other hand, acetate, propionate, butyrate, succinate and formate, the fermentation products the facultative anaerobes are significantly prevalent in PLA. The NMR based metabolic profile of ALA and PLA are evaluated taking polymerase chain reaction (PCR) and bacterial culture as gold standard method. However, when NMR results were compared with culture and PCR methods, a correct diagnosis of 94.11% in ALA (n = 85) and 100% in PLA (n = 10) cases were observed. NMR spectroscopy in conjunction with PCR and culture can expedite in differentiating ALA from PLA.