S. Ramaswamy, Ph.D.
CEO of c-CAMP, Dean, inStem, NCBS, Bangalore, India
Discovery, engineering and applications of Blue Fish Protein with Red Fluorescence
Swagatha Ghosh, Chi-Li Yu, Daniel Ferraro, Sai Sudha, Wayne Schaefer, David T Gibson and S. Ramaswamy
Fluorescent proteins and their applications have revolutionized our understanding of biology significantly. In spite of several years since the discovery of the classic GFP, proteins of this class are used as the standard flag bearers. We have recently discovered a protein from the fish Sanders vitrius that shows interesting fluorescent properties – including a 280 nm stoke shift and infrared emission. The crystal structure of the wild type protein shows that it is a tetramer. We have engineered mutations to make a monomer with very similar fluorescent properties. We have used this protein for tissue imaging as well as for in cell-fluorescence successfully
Gillian Murphy, Ph.D.
Professor, Department of Oncology, University of Cambridge, UK
A novel strategy for targeting metalloproteinases in cancer
Epithelial tumours evolve in a multi-step manner, involving both inflammatory and mesenchymal cells. Although intrinsic factors drive malignant progression, the influence of the micro-environment of neoplastic cells is a major feature of tumorigenesis. Extracellular proteinases, notably the metalloproteinases, are key players in the regulation of this cellular environment, acting as major effectors of both cell-cell and cell-extracellular matrix (ECM) interactions. They are involved in modifying ECM integrity, growth factor availability and the function of cell surface signalling systems, with consequent effects on cellular differentiation, proliferation and apoptosis.This has made metalloproteinases important targets for therapeutic interventions in cancer and small molecule inhibitors focussed on chelation of the active site zinc and binding within the immediate active site pocket were developed. These were not successful in early clinical trials due to the relative lack of specificity and precise knowledge of the target proteinase(s) in specific cancers. We can now appreciate that it is essential that we understand the relative roles of the different enzymes (of which there are over 60) in terms of their pro and anti tumour activity and their precise sites of expression The next generations of metalloproteinase inhibitors need the added specificity that might be gained from an understanding of the structure of individual active sites and the role of extra catalytic domains in substrate binding and other aspects of their biology. We have prepared scFv antibodies to the extra catalytic domains of two membrane metalloproteinases, MMP-14 and ADAM17, that play key roles in the tumour microenvironment. Our rationale and experiences with these agents will be presented in more detail.
Nader Pourmand, Ph.D.
Director, UCSC Genome Technology Center,University of California, Santa Cruz
Biosensor and Single Cell Manipulation using Nanopipettes
Approaching sub-cellular biological problems from an engineering perspective begs for the incorporation of electronic readouts. With their high sensitivity and low invasiveness, nanotechnology-based tools hold great promise for biochemical sensing and single-cell manipulation. During my talk I will discuss the incorporation of electrical measurements into nanopipette technology and present results showing the rapid and reversible response of these subcellular sensors to different analytes such as antigens, ions and carbohydrates. In addition, I will present the development of a single-cell manipulation platform that uses a nanopipette in a scanning ion-conductive microscopy technique. We use this newly developed technology to position the nanopipette with nanoscale precision, and to inject and/or aspirate a minute amount of material to and from individual cells or organelle without comprising cell viability. Furthermore, if time permits, I will show our strategy for a new, single-cell DNA/ RNA sequencing technology that will potentially use nanopipette technology to analyze the minute amount of aspirated cellular material.
Shigeki Miyamoto, Ph.D.
Professor, McArdle Laboratory for Cancer Research – UW Carbone Cancer Center
Department of Oncology, School of Medicine and Public Health
University of Wisconsin-Madison
“Inside-out” NF-κB signaling in cancer and other pathologies
The NF-κB/Rel family of transcription factors contributes to critical cellular processes, including immune, inflammatory and cell survival responses. As such, NF-κB is implicated in immunity-related diseases, as well as multiple types of human malignancies. Indeed, genetic alterations in the NF-κB signaling pathway are frequently observed in multiple human malignancies. NF-κB is normally kept inactive in the cytoplasm by inhibitor proteins. Extracellular ligands can induce the release of NF-κB from the inhibitors to allow its migration into the nucleus to regulate a variety of target genes. NF-κB activation is also induced in response to multiple stress conditions, including those induced by DNA-damaging anticancer agents. Although precise mechanisms are still unclear, research from our group has revealed a unique nuclear-to-cytoplasmic signaling pathway. In collaboration with bioengineers, clinicians and pharmaceutical industry, our lab has developed new methods to analyze primary cancer patient samples and identified several compounds with different mechanisms that mitigate this cell survival pathway. Further contributions from other labs have also revealed additional mechanisms and molecular players in this “inside-out” signaling pathway and expanded its role in other physiological and pathological processes, including B cell development, premature aging and therapy resistance of certain cancers. Our own new findings, along with these recent developments in the field, will be highlighted.
Sharmila Mande, Ph.D.
Principal Scientist and Head, Bio Sciences R&D, TCS Innovation Labs, Pune
Gut microbiome and health: Moving towards the new era of translational medicine
The microbes inhabiting our body outnumber our own cells by a factor of 10. The genomes of these microbes, called the ‘second genome’ are therefore expected to have great influence on our health and well being. The emerging field of metagenomics is rapidly becoming the method of choice for studying the microbial community (called microbiomes) present in various parts of the human body. Recent studies have implicated the role of gut microbiomes in several diseases and disorders. Studies have indicated gut microbiome’s role in nutrient absorption, immuno-modulation motor-response, and other key physiological processes. However, our understanding of the role of gut microbiota in malnutrition is currently incomplete. Exploration of these aspects are likely to help in understanding the microbial basis for several physiological disorders associated with malnutrition (eg, increased susceptibility to diarrhoeal pathogens) and may finally aid in devising appropriate probiotic strategies addressing this menace. A metagenomic approach was employed for analysing the differences between gut microbial communities obtained from malnourished and healthy children. Results of the analysis using TCS’ ‘Metagenomic Analysis Platform’ were discussed in detail during my talk.
Satheesh Babu T. G., Ph.D.
Associate Professor, Department of Sciences, School of Engineering, Amrita University, Coimbatore, India
Nanomaterials for ‘enzyme-free’ biosensing
Enzyme based sensors have many draw backs such as poor storage stability, easily affected by the change in pH and temperature and involves complicated enzyme immobilization procedures. To address this limitation, an alternative approach without the use of enzyme, “non-enzymatic” has been tried recently. Choosing the right catalyst for direct electrochemical oxidation / reduction of a target molecule is the key step in the fabrication of non-enzymatic sensors.
Non-enzymatic sensors for glucose, creatinine, vitamins and cholesterol are fabricated using different nanomaterials, such as nanotubes, nanowires and nanoparticles of copper oxide, titanium dioxide, tantalum oxide, platinum, gold and graphenes. These sensors selectively catalyse the targeted analyte with very high sensitivity. These nanomaterials based sensors combat the drawbacks of enzymatic sensors.
Gokul Das, Ph.D.
Co-Director, Breast Disease Site Research Group, Roswell Park Cancer Institute, Buffalo, NY
Probing Estrogen Receptor−Tumor Suppressor p53 Interaction in Cancer: From Basic Research to Clinical Trial
Tumor suppressor p53 and estrogen receptor have opposite roles in the onset and progression of breast cancer. p53 responds to a variety of cellular of stresses by restricting the proliferation and survival of abnormal cells. Estrogen receptor plays an important role in normal mammary gland development and the preservation of adult mammary gland function; however, when deregulated it becomes abnormally pro-proliferative and greatly contributes to breast tumorigenesis. The biological actions of estrogens are mediated by two genetically distinct estrogen receptors (ERs): ER alpha and ER beta. In addition to its expression in several ER alpha-positive breast cancers and normal mammary cells, ER beta is usually present in ER alpha-negative cancers including triple-negative breast cancer. In spite of genetically being wild type, why p53 is functionally debilitated in breast cancer has remained unclear. Our recent finding that ER alpha binds directly to p53 and inhibits its function has provided a novel mechanism for inactivating genetically wild type p53 in human cancer. Using a combination of proliferation and apoptosis assays, RNAi technology, quantitative chromatin immunoprecipitation (qChIP), and quantitative real-time PCR (qRT-PCR), in situ proximity ligation assay (PLA), and protein expression analysis in patient tissue micro array (TMA), we have demonstrated binding of ER alpha to p53 and have delineated the domains on both the proteins necessary for the interaction. Importantly, ionizing radiation inhibits the ER-p53 interaction in vivo both in human cancer cells and human breast tumor xenografts in mice. In addition, antiestrogenstamoxifen and faslodex/fulvestrant (ICI 182780) disrupt the ER-p53 interaction and counteract the repressive effect of ER alpha on p53, whereas 17β-estradiol (E2) enhances the interaction. Intriguingly, E2 has diametrically opposite effects on corepressor recruitment to a p53-target gene promoter versus a prototypic ERE-containing promoter. Thus, we have uncovered a novel mechanism by which estrogen could be providing a strong proliferative advantage to cells by dual mechanisms: enhancing expression of ERE-containing pro-proliferative genes while at the same time inhibiting transcription of p53-dependent anti-proliferative genes. Consistently, ER alpha enhances cell cycle progression and inhibits apoptosis of breast cancer cells. Correlating with these observations, our retrospective clinical study shows that presence of wild type p53 in ER-positive breast tumors is associated with better response to tamoxifen therapy. These data suggest ER alpha-p53 interaction could be one of the mechanisms underlying resistance to tamoxifen therapy, a major clinical challenge encountered in breast cancer patients. We have launched a prospective clinical trial to analyze ER-p53 interaction in breast cancer patient tumors at Roswell Park Cancer Institute. Our more recent finding that ER beta has opposite functions depending on the mutational status of p53 in breast cancer cells is significant in understanding the hard-to-treat triple-negative breast cancer and in developing novel therapeutic strategies against it. Our integrated approach to analyze ER-p53 interaction at the basic, translational, and clinical research levels has major implications in the diagnosis, prognosis, and treatment of breast cancer.
![Delegate Talk: Novel Cell-Based Biosensors for High Throughput Toxin Detection and Drug Screening Applications @ Amriteshwari Hall | Vallikavu | Kerala | India](http://www.amritabioquest.org/conference/2013/wp-content/uploads/sites/2/2015/02/biomed.jpg)
Anupama Natarajan, James Hickman and Peter Molnar
Novel Cell-Based Biosensors for High Throughput Toxin Detection and Drug Screening Applications
Over the last decade there has been focus on the development of cellbased biosensors to detect environmental toxins or to combat the threats of biological warfare. These sensors have been shown to have multiple applications including understanding function and behaviour at the cellular and tissue levels, in cell electrophysiology and as drug screening tools that can eliminate animal testing. These factors make the development of cell-based biosensors into high throughput systems a priority in pharmacological, environmental and defence industries (Pancrazio J J et al. 1999, Kang G et al. 2009, Krinke D et al. 2009). We have developed a high through-put in vitro cell-silicon hybrid platform that could be used to analyze both cell function and response to various toxins and drugs. Our hypothesis was that by utilizing surface modification to provide external guidance cues as well as optimal growth conditions for different cell types (Cardiac and Neuronal), we could enhance the information output and content of such a system. An intrinsic part of this study was to create ordered or patterned functional networks of cells on Micro-electrode arrays (MEA). Such engineered networks had a two-fold purpose in that they not only aided in a more accurate analysis of cell response and cell and tissue behaviour, but also increased the efficiency of the system by increasing the connectivity and placement of the cells over the recording electrodes. Here we show the response of this system to various toxins and drugs and the measurement of several vital cardiac parameters like conduction velocity and refractory period (Natarajan A et al. 2011)
Shrikant Anant, Ph.D.
The Department of Molecular & Integrative Physiology, Kansas University Medical Center, USA
Cancer Stem Cells: Target Colon Cancers
Shrikant Anant, Deep Kwatra and Dharmalingam Subramaniam
Colon cancer is a leading cause of cancer related deaths in the US, and its rate is increasing at an alarming rate in lndia. Recent studies have suggested the drug resistance role for a mall number of cells within a tumor called cancer stem cells. We identified the colon cancer stem cell marker DCLK1, a member of the protein kinase superfamily and the doublecortin family. The protein encodes a Cterminal serinethreonine protein kinase domain, which shows substantial homology to Ca2calmodulindependent protein kinase. Our current studies have been to identify compounds that can either affect DCLK1 expression or inhibits its activity as a way to inhibit cancer stem cells. Honokiol is a biphenolic compound that has been used in the traditional Chinese Medicine for treating various ailments. In vitro kinase assays with recombinant DCLK1 demonstrated that honokiol inhibits its kinase activity in a dose dependent manner. We therefore determined the effect of honokiol on stem cells. One method to look at effects on stem cells is perform a spheroid assay, where spheroids formation is suggested to maintain stemlike characteristic of cancer cells. Honokiol significantly suppressed colonosphere formation of two colon cancer cell lines HCT116 and SW480. Flow cytometry studies confirmed that honokiol reduced the number of DCLK1cells. A critical signaling pathway known to modulate intestinal stem cell proliferation is the Hippo signaling pathway, and deregulation of the pathway leads to tumor development. DCLK1cells had high levels of YAP1, the nuclear target of Hippo signaling. We determined the effect of honokiol on components of the hipposignaling pathway. Honokiol reduced the phosphorylation of Mst1/2, Lats1/2 and YAP1. Furthermore, honokiol treatment resulted in downregulation of YAPTEAD complex protein TEAD-1. Ectopic expression of the TEAD-1 partially rescued the cells from honokiol mediated growth suppression. To determine the effect of honokiol on tumor growth in vivo, nude mice harboring HCT116 tumor xenografts in their flanks were administered the compound intraperitoneally every day for 21 days. Honokiol treatment significantly inhibited tumor xenograft growth. Western blot and immunohistochemistry analyses demonstrated significant inhibition in the expression of stem marker and Hippo signaling proteins in the honokioltreated xenograft tissues. Taken together, these data suggest that honokiol is a potent inhibitor of colon cancer that targets DCLK1 stem cells by inhibiting Hippo signaling pathway.
![Delegate Talk: Inefficient NETosis: Cause for Predisposition to Recurrent Infections in Type 2 Diabetes @ Acharya Hall | Vallikavu | Kerala | India](http://www.amritabioquest.org/conference/2013/wp-content/uploads/sites/2/2015/02/gtm.jpg)
Manjunath Joshi, Apoorva Lad, Bharat Prasad Alevoor, Aswath Balakrishnan, Lingadakai Ramachandra and Kapaettu Satyamoorthy
Pathological conditions during Type 2 Diabetes (T2D) are associated with elevated risk for common community acquired infections due to poor glycemic control. Multiple studies have indicated specific defects in innate and adaptive immune function in diabetic subjects. Neutrophils play an important role in eliminating pathogens as an active constituent of innate immune system. Apart from canonically known phagocytosis mechanism, neutrophils are endowed with a unique ability to produce extracellular traps (NETs) to kill pathogens by expelling DNA coated with bactericidal proteins and histone. NETosis is stimulated by diverse bacteria and their products, fungi, protozoans, cytokines, phorbol esters and by activated platelets. Considering deregulation of metabolic and immune response pathways during pathological state of diabetes and NETosis as a potential mechanism for killing bacteria, we therefore, investigated whether hyperglycemic conditions modulate formation of neutrophil NETs and attempted to identify underlying immunoregulatory mechanisms. Freshly isolated neutrophils from normal individuals were cultured in absence or presence of high glucose (different concentrations) for 24 hours and activated with either LPS (2 mg/ml) or PMA (20 ng/ml) or IL-6 (20 ng/ml) for 3 hours. NETs were visualized and quantified by addition of DNA binding dye SYTOX green using fluorescence microscope and fluorimetry. NETs were quantified in Normal and diabetic subjects. Serum IL-6 levels were measured using ELISA technique. NETs bound elasatse were quantified in normal and diabetic subjects in presence or absence of DNase. Bacterial killing assays were performed upon infecting E.coli with activated neutrophils from normal and diabetic subjects. Microscopy and fluorimetry analysis suggested dramatic impairment in NETs formation under high glucose conditions. Extracellular DNA lattices formed in hyperglycemic conditions were short lived and unstable leading to rapid disintegration. Subsequent, time course experiments showed that NETs production was delayed in hyperglycemic conditions. To validate our findings more closely to clinical conditions, we investigated the neutrophil activation and NETs formation in diabetic patients. Upon stimulation with LPS for three hours, neutrophils from diabetic subjects responded weakly to LPS and lesser NETs were formed; whereas, neutrophils from normal individuals showed robust release of NETs. In few patients we found short and imperfect NETs in basal conditions suggesting constitutive activation of neutrophils in diabetic subjects. Interestingly, NETs bound elastase activity was reduced in diabetes subjects when compared to non-diabetic individuals, indicating a dysfunction of one of the important protein component of NETs during diabetes. Neutrophils from diabetic subjects released higher levels of IL-6 without any stimulation suggesting an existence of constitutively activated pro-inflammatory state. IL-6 induced NETs formation and was abrogated by high glucose. Weobserved that glycolysis inhibitor 2-DG resensitize the high glucose attenuated LPS and IL-6 induced NETs. a) NETs are influenced by glucose homeostasis, b) IL-6 as potent inducer of energy dependent NETs formation and c) hyperglycemia mimics a state of constitutively active pro-inflammatory condition in neutrophils leading to reduced response to external stimuli making diabetic subjects susceptible for infections.
![Delegate Talk: A Non-toxic Approach to Prevent Secondary Effects of Chemotherapies @ Acharya Hall | Vallikavu | Kerala | India](http://www.amritabioquest.org/conference/2013/wp-content/uploads/sites/2/2015/02/gtm.jpg)
Tanu Sharma, Narendra Parihar, Someshwar Nath, Azad Singh, Savneet Kaur, Simendra Singh and Chandi Mandal.
In the recent years, there has been a surge in various forms of cancers especially in urban areas of India. Tumor recurrence and metastasis are the main causes of increased morbidity and mortality. In poor and developing countries like India chemotherapy is the choosen method of therapeutic intervention for different types of malignancies such as breast, prostate, pancreas, etc. Although general chemotherapy treatment is effective in controlling tumour growth but high doses of the treatment regimen often results in severe off-target toxicity to different organs including liver, kidney, heart, brain etc. (1) A large percentage of patients are unable to tolerate this toxicity and sometime it can also lead to life threatening complications. (2) Therefore, the primary objective of this study is to prevent this druginduced off-target toxicity. Omega 3 fatty acids (n-3FAs) such as DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid) that are active components of fish oil play active role in preventing cancer growth and its metastasis. (3–5) We have recently identified that omega 3-fatty acids prevent breast cancer bone metastasis by targeting cancer stem cell marker CD44 and osteoclastogenic factor CSF-1(4, 5). With this background, the present study addresses the role of n-3FAs in preventing chemotherapeutic drug-induced off-target toxicity.
Methods
The cat fishes were divided into four groups (each group contains 4 fishes) for each set of experiments. Three different chemotherapeutic drugs etoposide, doxorubicin, cisplatin at higher dosages (4, 2, 2 mg/kg body wt respectively) and fish oil (60mg/kg body wt, DHA: EPA; 2:3) were administered to cat fish (Mangur) by gavage. After 6 days, cat fishes were sacrificed and different organs were isolated. SGPT and alkaline phosphatase (ALP) activity assays were performed to examine liver function and superoxide dismutase activity (SOD) assay was conducted to evaluate reactive oxygen species (ROS) level.
Results
Severe skin damages were observed both in doxorubicin and cisplatintreated cat fishes as compared to the control fishes, indicating side effects of chemotherapeutic drugs treatment. High level of SGPT as well as ALP activity was observed in liver samples of etoposide and doxorubicin – treated fishes, suggesting chemotherapeutic drug-induced liver toxicity. Mechanistically, we found that doxorubicin treatment showed significant decrease of SOD activity in liver samples in comparison to the control fishes, suggesting that chemotherapeutic drugs lead to organ toxicity presumably by increasing ROS levels (6). However, treatment of the fishes with n-3FAs of the fish oil led to a dramatic reduction of chemotherapeutic drug-induced skin damages. Also, low levels of SGPT and ALP activities were observed in the fishes given etoposide in combination with n-3FAs as compared to the fishes given etoposide alone. Similarly, fish oil also gave protection against doxorubicin-induced liver dysfunction. Our data further showed that n-3FAs treatment significantly increased etoposide and doxorubicin-inhibited SOD activity.
Conclusions
The study for the first time reports that the use of n-3FAs leads to a dramatic reduction of chemotherapeutic drug-induced skin damages and mitigates chemotherapeutic drug-driven liver dysfunction presumably by reducing ROS level. This study suggests that n-3FAs may possibly be used in combination with chemotherapeutic drugs to treat different cancers to reduce chemotherapeutic drugs-associated systemic toxicity and to increase anticancer activity. Ongoing research study will further address the role of n-3FAs on other off-target toxic effects of chemotherapeutic drugs.
Jeff Perry, Ph.D.
Assistant Professor, University of California, Riverside
Combined Crystallography and SAXS Methods for Studying Macromolecular Complexes
Recent developments in small angle X-ray scattering (SAXS) are rapidly providing new insights into protein interactions, complexes and conformational states in solution, allowing for detailed biophysical quantification of samples of interest1. Initial analyses provide a judgment of sample quality, revealing the potential presence of aggregation, the overall extent of folding or disorder, the radius of gyration, maximum particle dimensions and oligomerization state. Structural characterizations may include ab initio approaches from SAXS data alone, or enhance structural solutions when combined with previously determined crystal/NMR domains. This combination can provide definitions of architectures, spatial organizations of the protein domains within a complex, including those not yet determined by crystallography or NMR, as well as defining key conformational states. Advantageously, SAXS is not generally constrained by macromolecule size, and rapid collection of data in a 96-well plate format provides methods to screen sample conditions. Such screens include co-factors, substrates, differing protein or nucleotide partners or small molecule inhibitors, to more fully characterize the variations within assembly states and key conformational changes. These analyses are also useful for screening constructs and conditions that are most likely to promote crystal growth. Moreover, these high throughput structural determinations can be leveraged to define how polymorphisms affect assembly formations and activities. Also, SAXS-based technologies may be potentially used for novel structure-based screening, for compounds inducing shape changes or associations/diassociations. This is addition to defining architectural characterizations of complexes and interactions for systems biology-based research, and distinctions in assemblies and interactions in comparative genomics. Thus, SAXS combined with crystallography/NMR and computation provides a unique set of tools that should be considered as being part of one’s repertoire of biophysical analyses, when conducting characterizations of protein and other macromolecular interactions.
1 Perry JJ & Tainer JA. Developing advanced X-ray scattering methods combined with crystallography and computation. Methods. 2013 Mar;59(3):363-71.
![Delegate Talk: Proteomic profiling of gallbladder cancer secretome – a source for circulatory biomarker discovery @ Amriteshwari Hall | Vallikavu | Kerala | India](http://www.amritabioquest.org/conference/2013/wp-content/uploads/sites/2/2015/02/bioanalt.jpg)
Tejaswini Subbannayya, Nandini A. Sahasrabuddhe, Arivusudar Marimuthu, Santosh Renuse, Gajanan Sathe, Srinivas M. Srikanth, Mustafa A. Barbhuiya, Bipin Nair, Juan Carlos Roa, Rafael Guerrero-Preston, H. C. Harsha, David Sidransky, Akhilesh Pandey, T. S. Keshava Prasad and Aditi Chatterjee
Proteomic profiling of gallbladder cancer secretome – a source for circulatory biomarker discovery
Gallbladder cancer (GBC) is the fifth most common cancer of the gastrointestinal tract and one of the common malignancies that occur in the biliary tract (Misra et al. 2006; Lazcano-Ponce et al. 2001). It has a poor prognosis with survival of less than 5 years in 90% of the cases (Misra et al. 2003). The etiology is ill-defined. Several risk factors have been reported including cholelithiasis, obesity, female gender and exposure to carcinogens (Eslick 2010; Kumar et al. 2006). Poor prognosis in GBC is mainly due to late presentation of the disease and lack of reliable biomarkers for early diagnosis. This emphasizes the need to identify and characterize cancer biomarkers to aid in the diagnosis and prognosis of GBC. Secreted proteins are an important class of molecules which can be detected in body fluids and has been targeted for biomarker discovery. There are challenges faced in the proteomic interrogation of body fluids especially plasma such as low abundance of tumor secreted proteins, high complexity and high abundance of other proteins that are not released by the tumor cells (Tonack et al. 2009). Profiling of conditioned media from the cancer cell lines can be used as an alternate means to identify secreted proteins from tumor cells (Kashyap et al. 2010; Marimuthu et al. 2012). We analyzed the invasive property of 7 GBC cell lines (SNU-308, G-415, GB-d1, TGBC2TKB, TGBC24TKB, OCUG-1 and NOZ). Four cell lines were selected for analysis of the cancer secretome based on the invasive property of the cells. We employed isobaric tags for relative and absolute quantitation (iTRAQ) labeling technology coupled with high resolution mass spectrometry to identify and characterize secretome from the panel of 4GBC cancer cells mentioned above. In total, we have identified around 2,000 proteins of which 175 were secreted at differential abundance across all the four cell lines. This secretome analysis will act as a reservoir of candidate biomarkers. Currently, we are investigating and validating these candidate markers from GBC cell secretome. Through this study, we have shown mass spectrometry-based quantitative proteomic analysis as a robust approach to investigate secreted proteins in cancer cells.