Papers by Srabani Bhaumik
Proceedings of the National Academy of Sciences of the United States of America, Dec 18, 2001
Imaging reporter gene expression in living subjects is a rapidly evolving area of molecular imagi... more Imaging reporter gene expression in living subjects is a rapidly evolving area of molecular imaging research. Studies have validated the use of reporter genes with positron emission tomography (PET), single photon emission computed tomography (SPECT), MRI, fluorescence with wild-type and mutants of green fluorescent protein, as well as bioluminescence using Firefly luciferase en-zyme͞protein (FL). In the current study, we validate for the first time the ability to image bioluminescence from Renilla luciferase enzyme͞protein (RL) by injecting the substrate coelenterazine in living mice. A highly sensitive cooled charge-coupled device camera provides images within a few minutes of photon counting. Cells, transiently expressing the Rluc were imaged while located in the peritoneum, s.c. layer, as well as in the liver and lungs of living mice tail-vein injected with coelenterazine. Furthermore, D-luciferin (a substrate for FL) does not serve as a substrate for RL, and coelenterazine does not serve as a substrate for FL either in cell culture or in living mice. We also show that both Rluc and Fluc expression can be imaged in the same living mouse and that the kinetics of light production are distinct. The approaches validated will have direct applications to various studies where two molecular events need to be tracked, including cell trafficking of two cell populations, two gene therapy vectors, and indirect monitoring of two endogenous genes through the use of two reporter genes.
Proceedings of the National Academy of Sciences, 2004
Spliceosome-mediated RNA trans-splicing (SMaRT) provides an effective means to reprogram mRNAs an... more Spliceosome-mediated RNA trans-splicing (SMaRT) provides an effective means to reprogram mRNAs and the proteins they encode. SMaRT technology has a broad range of applications, including RNA repair and molecular imaging, each governed by the nature of the sequences delivered by the pre-trans-splicing molecule. Here, we show the ability of SMaRT to optically image the expression of an exogenous gene at the level of pre-mRNA splicing in cells and living animals. Because of the modular design of pre-trans-splicing molecules, there is great potential to employ SMaRT to image the expression of any arbitrary gene of interest in living subjects. In this report, we describe a model system that demonstrates the feasibility of imaging gene expression by transsplicing in small animals. This represents a previously undescribed approach to molecular imaging of mRNA levels in living subjects.
Gene Therapy, 2011
Gene-directed enzyme prodrug therapy (GDEPT) is a promising and emerging strategy that attempts t... more Gene-directed enzyme prodrug therapy (GDEPT) is a promising and emerging strategy that attempts to limit the systemic toxicity inherent to cancer chemotherapy by means of tumor-targeted delivery and expression of an exogenous gene whose product converts nontoxic prodrug(s) into activated cytotoxic agent(s). The bacterial nitroreductase (NTR) enzyme, coupled with its substrate prodrug 5-(azaridin-1-yl)-2,4-dinitrobenzamide (CB1954), is a promising GDEPT strategy that has reached clinical trials. However, no strategy exists to visually monitor and quantitatively evaluate the therapeutic efficacy of NTR/CB1954 prodrug therapy in cells and imaging in living animals. As the success of any GDEPT is dependent upon the efficiency of transgene expression in vivo, we developed a safe, sensitive and reproducible noninvasive imaging method to monitor NTR transgene expression that would allow quantitative assessment of both therapeutic efficacy and diagnostic outcome of NTR/ CB1954 prodrug therapy in the future. Here, we investigate the use of a novel fluorescent imaging dye CytoCy5S (a Cy5-labeled quenched substrate of NTR enzyme) on various cancer cell lines in vitro and in NTR-transfected tumor-bearing animals in vivo. CytoCy5S-labeled cells become fluorescent at 'red-shifted' wavelengths (638 nm) when reduced by cellular NTR enzyme and remains trapped within the cells for extended periods of time. The conversion and entrapment was dynamically recorded using a time-lapsed microscopy. Systemic and intratumoral delivery of CytoCy5S to NTR-expressing tumors in animals indicated steady and reproducible signals even 16 h after delivery (Po0.001). This is the first study to address visual monitoring and quantitative evaluation of NTR activity in small animals using CytoCy5S, and establishes the capability of NTR to function as an imageable reporter gene.
Radiology, Sep 22, 2016
Purpose To quantitatively determine the limit of detection of marrow stromal cells (MSC) after ca... more Purpose To quantitatively determine the limit of detection of marrow stromal cells (MSC) after cardiac cell therapy (CCT) in swine by using clinical positron emission tomography (PET) reporter gene imaging and magnetic resonance (MR) imaging with cell prelabeling. Materials and Methods Animal studies were approved by the institutional administrative panel on laboratory animal care. Seven swine received 23 intracardiac cell injections that contained control MSC and cell mixtures of MSC expressing a multimodality triple fusion (TF) reporter gene (MSC-TF) and bearing superparamagnetic iron oxide nanoparticles (NP) (MSC-TF-NP) or NP alone. Clinical MR imaging and PET reporter gene molecular imaging were performed after intravenous injection of the radiotracer fluorine 18-radiolabeled 9-[4-fluoro-3-(hydroxyl methyl) butyl] guanine ((18)F-FHBG). Linear regression analysis of both MR imaging and PET data and nonlinear regression analysis of PET data were performed, accounting for multiple ...
Journal of Cellular Biochemistry, 2020
In preclinical cancer studies, three-dimensional (3D) cell spheroids and aggregates are preferred... more In preclinical cancer studies, three-dimensional (3D) cell spheroids and aggregates are preferred over monolayer cell cultures due to their architectural and functional similarity to solid tumors. We performed a proof-of-concept study to generate physiologically relevant and predictive preclinical models using non-small cell lung adenocarcinoma, and colon and colorectal adenocarcinoma cell line-derived 3D spheroids and aggregates. Distinct panels were designed to determine the expression profiles of frequently studied biomarkers of the two cancer subtypes. The lung adenocarcinoma panel included ALK, EGFR, TTF-1, and CK7 biomarkers, and the colon and colorectal adenocarcinoma panel included BRAF V600E, MSH2, MSH6, and CK20. Recent advances in immunofluorescence (IF) multiplexing and imaging technology enable simultaneous detection and quantification of multiple biomarkers on a single slide. In this study, we performed IF staining of multiple biomarkers per section on formalin-fixed paraffin-embedded 3D spheroids and aggregates. We optimized protocol parameters for automated IF and demonstrated staining concordance with automated chromogenic immunohistochemistry performed with validated protocols. Next, post-acquisition spectral unmixing of the captured fluorescent signals were utilized to delineate four differently stained biomarkers within a single multiplex IF image, followed by automated quantification of the expressed markers. This workflow has the potential to be adapted to preclinical high-throughput screening and drug efficacy studies utilizing 3D spheroids from cancer cell lines and patient-derived organoids. The process allows for cost, time, and resource savings through concurrent staining of several biomarkers on a single slide, the ability to study the
Journal for ImmunoTherapy of Cancer
Since the publication of the Society for Immunotherapy of Cancer’s (SITC) original cancer immunot... more Since the publication of the Society for Immunotherapy of Cancer’s (SITC) original cancer immunotherapy biomarkers resource document, there have been remarkable breakthroughs in cancer immunotherapy, in particular the development and approval of immune checkpoint inhibitors, engineered cellular therapies, and tumor vaccines to unleash antitumor immune activity. The most notable feature of these breakthroughs is the achievement of durable clinical responses in some patients, enabling long-term survival. These durable responses have been noted in tumor types that were not previously considered immunotherapy-sensitive, suggesting that all patients with cancer may have the potential to benefit from immunotherapy. However, a persistent challenge in the field is the fact that only a minority of patients respond to immunotherapy, especially those therapies that rely on endogenous immune activation such as checkpoint inhibitors and vaccination due to the complex and heterogeneous immune esc...
Laboratory investigation; a journal of technical methods and pathology, Jul 15, 2017
The ability to simultaneously visualize the presence, abundance, location and functional state of... more The ability to simultaneously visualize the presence, abundance, location and functional state of many targets in cells and tissues has been described as a true next-generation approach in immunohistochemistry (IHC). A typical requirement for multiplex IHC (mIHC) is the use of different animal species for each primary (1°Ab) and secondary (2°Ab) antibody pair. Although 1°Abs from different species have been used with differently labeled species-specific 2°Abs, quite often the appropriate combination of antibodies is not available. More recently, sequential detection of multiple antigens using 1°Abs from the same species used a microwaving treatment between successive antigen detection cycles to elute previously bound 1°Ab/2°Ab complex and therefore to prevent the cross-reactivity of anti-species 2°Abs used in subsequent detection cycles. We present here a fully automated 1°Ab/2°Ab complex heat deactivation (HD) method on Ventana's BenchMark ULTRA slide stainer. This method is ap...
Journal for ImmunoTherapy of Cancer, 2016
P190 Multi-genome reassortant dendritic cell-tropic vector platform (ZVex®-Multi) allows flexible... more P190 Multi-genome reassortant dendritic cell-tropic vector platform (ZVex®-Multi) allows flexible co-expression of multiple antigens and immune modulators for optimal induction of anti-tumor CD8+ T cell responses
Eur J Nucl Med Mol Imaging, 2008
Purpose-Firefly luciferase catalyzes the oxidative decarboxylation of D-luciferin to oxyluciferin... more Purpose-Firefly luciferase catalyzes the oxidative decarboxylation of D-luciferin to oxyluciferin in the presence of cofactors, producing bioluminescence. This reaction is used in optical bioluminescence-based molecular imaging approaches to detect the expression of the firefly luciferase reporter gene. Biokinetics and distribution of the substrate most likely have a significant impact on levels of light signal and therefore need to be investigated. Methods-Benzene ring 14 C(U)-labeled D-luciferin was utilized. Cell uptake and efflux assays, murine biodistribution, autoradiography and CCD-camera based optical bioluminescence imaging were carried out to examine the in vitro and in vivo characteristics of the tracer in cell culture and in living mice respectively. Results-Radiolabeled and unlabeled D-luciferin revealed comparable levels of light emission when incubated with equivalent amounts of the firefly luciferase enzyme. Cell uptake assays in pCMV-luciferase-transfected cells showed slow trapping of the tracer and relatively low uptake values (up to 22.9-fold higher in firefly luciferase gene-transfected vs. nontransfected cells,
ABSTRACT Imaging cells after cardiac cellular therapy could be a promising approach to optimizing... more ABSTRACT Imaging cells after cardiac cellular therapy could be a promising approach to optimizing this new treatment strategy. Marrying the strengths of cardiovascular MRI (high spatial resolution) and PET (high sensitivity) enhances the ability to assess the cell location, viability, survival, and myocardial response to treatment. To build on our previous swine studies with adenovirally transduced cells, rat mesenchymal stem cells (rMSC) were stably transduced with a second generation, self-inactivating lentivirus carrying a ubiquitin-driven triple fusion (TF) reporter gene (RG), comprised of green fluorescent protein (GFP), firefly luciferase (Fluc) and a mutant truncated herpes simplex virus type 1 thymidine kinase (HSV1-tsr39tk). In vitro studies indicated that sorting lentivirally transduced cells enhanced levels of TFRG levels, by sorting transduced cells that were 102 to 103 above background fluorescence. Cells were injected in vivo and demonstrated a linear relationship between when implanted subcutaneously between average radiance (photons/s/cm2 /sr) and cell number ( R2= 0.9682). The highest expressing cells demonstrated a 3.1 fold increase in average radiance (photons/s/cm2 /sr) from the initial cells after sorting three times and demonstrated a semi-logarithmic relationship, as expected (R2= 0.9864). The PET reporter gene activity of MSC-TF was assessed of 3H-penciclovir. At 3 hours activity was significantly higher than control C6 Rat glioma cells bearing the HSV-sr39ttk gene. For in vivo cell imaging studies, either all the MSC-TF cells, or 1/5 fraction of the total cells, were labeled with 30 µg/ml Iron bearing SPIO particles (35nm) using a protamine transfection technique, and injected into the hearts of swine after thoracotomy (n=4 swine). MRI was performed using the Fiesta or FGRE sequences allowed spatiotemporal distinction between cells and control particles, and allowed detection of cells after injection of 150-350 x106 cells. Next, clinical PET-CT was performed dynamically up to five hours after injection of F18-FHBG (14 mCi), and nine hours after injection of the rMSC-TF.. A linear relationship was determined between the number of cells injected and the SUV (standard uptake value) and signal to background ratio compared to cell number injected (n=3 swine). After 5 hours post injection, Mean SUV (Standard uptake value) and Signal/Background ratio was 0.063 and 2.11 for 150 x 106, 0.112 and 2.02 for 200 x 106, 0.189 and 2.34 for 250 x 106, and 0.227 and 2.82 for 300 x 106. Our results indicate that MRI and PET can be married together to localize injected cells for translational cell imaging. Future work will aim to use swine TFRG-MSC in survival models of swine infarction. Studies of PET reporter genes in large animals should help translate stem cells to the clinic and establish the role for molecular imaging in monitoring stem cell therapies.
PLOS ONE, 2015
Objective Ultra-small superparamagnetic iron oxide nanoparticles (USPIO) are promising contrast a... more Objective Ultra-small superparamagnetic iron oxide nanoparticles (USPIO) are promising contrast agents for magnetic resonance imaging (MRI). USPIO mediated proton relaxation rate enhancement is strongly dependent on compartmentalization of the agent and can vary depending on their intracellular or extracellular location in the tumor microenvironment. We compared the T1-and T2-enhancement pattern of intracellular and extracellular USPIO in mouse models of cancer and pilot data from patients. A better understanding of these MR signal effects will enable non-invasive characterizations of the composition of the tumor microenvironment.
Journal for ImmunoTherapy of Cancer, 2015
PURPOSE Tumor-associated macrophages (TAM) maintain a chronic inflammation in cancers, which is a... more PURPOSE Tumor-associated macrophages (TAM) maintain a chronic inflammation in cancers, which is associated with tumor aggressiveness and poor prognosis. As new anti-inflammatory therapeutics targeting TAM enter the clinic, it becomes increasingly important to identify patients whose tumors are heavily infiltrated by TAM. GEH121333 is a novel ultrasmall superparamagnetic iron oxide compound, currently developed for clinical applications. The purpose of this study was to (1) assess if GEH121333 can serve as a MR imaging biomarker for TAM, and (2) compare tumor MR enhancement profiles between GEH121333 and ferumoxytol. METHOD AND MATERIALS The study was approved by the institutional animal care and use committee. Mice harboring MMTV-PyMT-derived adenocarcinomas underwent serial MR scans before as well as directly after, 24, 48 and 72 hours (h) post-injection (p.i.) of GEH121333 (n=10) or ferumoxytol (n=9), using a 7T animal MR scanner and T1, T2 and T2*-weighted pulse sequences. Tumor ...
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Papers by Srabani Bhaumik