Longitudinal Monitoring of Antibody Responses against Tumor Cells Using Magneto-nanosensors with a Nanoliter of Blood.

Each immunoglobulin isotype has unique immune effector functions. The contribution of these functions in the elimination of pathogens and tumors can be determined by monitoring quantitative temporal changes in isotype levels. Here, we developed a novel technique using magneto-nanosensors based on the effect of giant magnetoresistance (GMR) for longitudinal monitoring of total and antigen-specific isotype levels with high precision, using as little as 1 nL of serum. Combining in vitro serologic measurements with in vivo imaging techniques, we investigated the role of the antibody response in the regression of firefly luciferase (FL)-labeled lymphoma cells in spleen, kidney, and lymph nodes in a syngeneic Burkitt's lymphoma mouse model. Regression status was determined by whole body bioluminescent imaging (BLI). The magneto-nanosensors revealed that anti-FL IgG2a and total IgG2a were elevated and sustained in regression mice compared to non-regression mice (p < 0.05). This platform shows promise for monitoring immunotherapy, vaccination, and autoimmunity.

Discovery and validation of small-molecule heat-shock protein 90 inhibitors through multimodality molecular imaging in living subjects.

Up-regulation of the folding machinery of the heat-shock protein 90 (Hsp90) chaperone protein is crucial for cancer progression. The two Hsp90 isoforms (α and ß) play different roles in response to chemotherapy. To identify isoform-selective inhibitors of Hsp90(α/ß)/cochaperone p23 interactions, we developed a dual-luciferase (Renilla and Firefly) reporter system for high-throughput screening (HTS) and monitoring the efficacy of Hsp90 inhibitors in cell culture and live mice. HTS of a 30,176 small-molecule chemical library in cell culture identified a compound, N-(5-methylisoxazol-3-yl)-2-[4-(thiophen-2-yl)-6-(trifluoromethyl)pyrimidin-2-ylthio]acetamide (CP9), that binds to Hsp90(α/ß) and displays characteristics of Hsp90 inhibitors, i.e., degradation of Hsp90 client proteins and inhibition of cell proliferation, glucose metabolism, and thymidine kinase activity, in multiple cancer cell lines. The efficacy of CP9 in disrupting Hsp90(α/ß)/p23 interactions and cell proliferation in tumor xenografts was evaluated by non-invasive, repetitive Renilla luciferase and Firefly luciferase imaging, respectively. At 38 h posttreatment (80 mg/kg × 3, i.p.), CP9 led to selective disruption of Hsp90α/p23 as compared with Hsp90ß/p23 interactions. Small-animal PET/CT in the same cohort of mice showed that CP9 treatment (43 h) led to a 40% decrease in (18)F-fluorodeoxyglucose uptake in tumors relative to carrier control-treated mice. However, CP9 did not lead to significant degradation of Hsp90 client proteins in tumors. We performed a structural activity relationship study with 62 analogs of CP9 and identified A17 as the lead compound that outperformed CP9 in inhibiting Hsp90(α/ß)/p23 interactions in cell culture. Our efforts demonstrated the power of coupling of HTS with multimodality molecular imaging and led to identification of Hsp90 inhibitors.

Bioluminescence resonance energy transfer (BRET) imaging of protein-protein interactions within deep tissues of living subjects.

Identifying protein-protein interactions (PPIs) is essential for understanding various disease mechanisms and developing new therapeutic approaches. Current methods for assaying cellular intermolecular interactions are mainly used for cells in culture and have limited use for the noninvasive assessment of small animal disease models. Here, we describe red light-emitting reporter systems based on bioluminescence resonance energy transfer (BRET) that allow for assaying PPIs both in cell culture and deep tissues of small animals. These BRET systems consist of the recently developed Renilla reniformis luciferase (RLuc) variants RLuc8 and RLuc8.6, used as BRET donors, combined with two red fluorescent proteins, TagRFP and TurboFP635, as BRET acceptors. In addition to the native coelenterazine luciferase substrate, we used the synthetic derivative coelenterazine-v, which further red-shifts the emission maxima of Renilla luciferases by 35 nm. We show the use of these BRET systems for ratiometric imaging of both cells in culture and deep-tissue small animal tumor models and validate their applicability for studying PPIs in mice in the context of rapamycin-induced FK506 binding protein 12 (FKBP12)-FKBP12 rapamycin binding domain (FRB) association. These red light-emitting BRET systems have great potential for investigating PPIs in the context of drug screening and target validation applications.

Noninvasive molecular imaging of c-Myc activation in living mice.

The cytoplasmic Myc protein (c-Myc) regulates various human genes and is dysregulated in many human cancers. Phosphorylation mediates the protein activation of c-Myc and is essential for the function of this transcription factor in normal cell behavior and tumor growth. To date, however, the targeting of Myc as a therapeutic approach for cancer treatment has been achieved primarily at the nonprotein level. We have developed a molecular imaging sensor for noninvasive imaging of c-Myc activity in living subjects using a split Firefly luciferase (FL) complementation strategy to detect and quantify the phosphorylation-mediated interaction between glycogen synthase kinase 3beta (GSK3beta) and c-Myc. This sensor system consists of two fusion proteins, GSK 35-433-CFL and NFL-c-Myc, in which specific fragments of GSK3beta and c-Myc are fused with C-terminal and N-terminal fragments of the split FL, respectively. The sensor detects phosphorylation-specific GSK3beta-c-Myc interaction, the imaging signal of which correlates with the steady-state and temporal regulation of c-Myc phosphorylation in cell culture. The sensor also detects inhibition of c-Myc activity via differential pathways, allowing noninvasive monitoring of c-Myc-targeted drug efficacy in intact cells and living mice. Notably, this drug inhibition is detected before changes in tumor size are apparent in mouse xenograft and liver tumor models. This reporter system not only provides an innovative way to investigate the role of functional c-Myc in normal and cancer-related biological processes, but also facilitates c-Myc-targeted drug development by providing a rapid quantitative approach to assessing cancer response to therapy in living subjects.

In Vivo Evaluation of Near-Infrared Fluorescent Probe for TIM3 Targeting in Mouse Glioma.

PURPOSE: Current checkpoint inhibitor immunotherapy strategies in glioblastoma are challenged by mechanisms of resistance including an immunosuppressive tumor microenvironment. T cell immunoglobulin domain and mucin domain 3 (TIM3) is a late-phase checkpoint receptor traditionally associated with T cell exhaustion. We apply fluorescent imaging techniques to explore feasibility of in vivo visualization of the immune state in a glioblastoma mouse model. PROCEDURES: TIM3 monoclonal antibody was conjugated to a near-infrared fluorescent dye, IRDye-800CW (800CW). The TIM3 experimental conjugate and isotype control were assessed for specificity with immunofluorescent staining and flow cytometry in murine cell lines (GL261 glioma and RAW264.7 macrophages). C57BL/6 mice with orthotopically implanted GL261 cells were imaged in vivo over 4 days after intravenous TIM3-800CW injection to assess tumor-specific uptake. Cell-specific uptake was then assessed on histologic sections. RESULTS: The experimental TIM3-800CW, but not its isotype control, bound to RAW264.7 macrophages in vitro. Specificity to RAW264.7 macrophages and not GL261 tumor cells was quantitatively confirmed with the corresponding clone of TIM3 on flow cytometry. In vivo fluorescence imaging of the 800CW signal was localized to the intracranial tumor and significantly higher for the TIM3-800CW cohort, relative to non-targeting isotype control, immediately after tail vein injection and for up to 48 h after injection. Resected organs of tumor bearing mice showed significantly higher uptake in the liver and spleen. TIM3-800CW was seen to co-stain with CD3 (13%), CD11b (29%), and CD206 (26%). CONCLUSIONS: We propose fluorescent imaging of immune cell imaging as a potential strategy for monitoring and localizing immunologically relevant foci in the setting of brain tumors. Alternative markers and target validation will further clarify the temporal relationship of immunosuppressive effector cells throughout glioma resistance.

A flexible electronic strain sensor for the real-time monitoring of tumor regression.

Assessing the efficacy of cancer therapeutics in mouse models is a critical step in treatment development. However, low-resolution measurement tools and small sample sizes make determining drug efficacy in vivo a difficult and time-intensive task. Here, we present a commercially scalable wearable electronic strain sensor that automates the in vivo testing of cancer therapeutics by continuously monitoring the micrometer-scale progression or regression of subcutaneously implanted tumors at the minute time scale. In two in vivo cancer mouse models, our sensor discerned differences in tumor volume dynamics between drug- and vehicle-treated tumors within 5 hours following therapy initiation. These short-term regression measurements were validated through histology, and caliper and bioluminescence measurements taken over weeklong treatment periods demonstrated the correlation with longer-term treatment response. We anticipate that real-time tumor regression datasets could help expedite and automate the process of screening cancer therapies in vivo.

A miniaturized optoelectronic biosensor for real-time point-of-care total protein analysis.

A miniaturized optoelectronic sensor is demonstrated that measures total protein concentration in serum and urine with sensitivity and accuracy comparable to gold-standard methods. The sensor is comprised of a vertical cavity surface emitting laser (VCSEL), photodetector and other custom optical components and electronics that can be hybrid packaged into a portable, handheld form factor. In conjunction, a custom fluorescence assay has been developed based on the protein-induced fluorescence enhancement (PIFE) phenomenon, enabling real-time sensor response to changes in protein concentration. Methods are described for the following:•Standard curves: Used to determine the sensitivity, dynamic range, and linearity of the VCSEL biosensor/PIFE assay system in buffer as well as in human blood and urine samples.•Comparison of VCSEL biosensor performance with a benchtop fluorimetric microplate reader.•Accuracy of the VCSEL biosensor/PIFE assay system: Evaluated by comparing sensor measurements with gold-standard clinical laboratory measurements of total protein in serum and urine samples from patients with diabetes.

Whole-body PET Imaging of T-cell Response to Glioblastoma.

PURPOSE: Immunotherapy is a promising approach for many oncological malignancies, including glioblastoma, however, there are currently no available tools or biomarkers to accurately assess whole-body immune responses in patients with glioblastoma treated with immunotherapy. Here, the utility of OX40, a costimulatory molecule mainly expressed on activated effector T cells known to play an important role in eliminating cancer cells, was evaluated as a PET imaging biomarker to quantify and track response to immunotherapy. EXPERIMENTAL DESIGN: A subcutaneous vaccination approach of CpG oligodeoxynucleotide, OX40 mAb, and tumor lysate at a remote site in a murine orthotopic glioma model was developed to induce activation of T cells distantly while monitoring their distribution in stimulated lymphoid organs with respect to observed therapeutic effects. To detect OX40-positive T cells, we utilized our in-house-developed 89Zr-DFO-OX40 mAb and in vivo PET/CT imaging. RESULTS: ImmunoPET with 89Zr-DFO-OX40 mAb revealed strong OX40-positive responses with high specificity, not only in the nearest lymph node from vaccinated area (mean, 20.8%ID/cc) but also in the spleen (16.7%ID/cc) and the tumor draining lymph node (11.4%ID/cc). When the tumor was small (<106 p/sec/cm2/sr in bioluminescence imaging), a high number of responders and percentage shrinkage in tumor signal was indicated after only a single cycle of vaccination. CONCLUSIONS: The results highlight the promise of clinically translating cancer vaccination as a potential glioma therapy, as well as the benefits of monitoring efficacy of these treatments using immunoPET imaging of T-cell activation.

Real-time point-of-care total protein measurement with a miniaturized optoelectronic biosensor and fast fluorescence-based assay.

Measurement of total protein in urine is key to monitoring kidney health in diabetes. However, most total protein assays are performed using large, expensive laboratory chemistry analyzers that are not amenable to point-of-care analysis or home monitoring and cannot provide real-time readouts. We developed a miniaturized optoelectronic biosensor using a vertical cavity surface-emitting laser (VCSEL), coupled with a fast protein assay based on protein-induced fluorescence enhancement (PIFE), that can dynamically measure protein concentrations in protein-spiked buffer, serum, and urine in seconds with excellent sensitivity (urine LOD = 0.023 g/L, LOQ = 0.075 g/L) and over a broad range of physiologically relevant concentrations. Comparison with gold standard clinical assays and standard fluorimetry tools showed that the sensor can accurately and reliably quantitate total protein in clinical urine samples from patients with diabetes. Our VCSEL biosensor is amenable to integration with miniaturized electronics, which could afford a portable, low-cost, easy-to-use device for sensitive, accurate, and real-time total protein measurements from small biofluid volumes.

Molecular imaging of a fluorescent antibody against epidermal growth factor receptor detects high-grade glioma.

The prognosis for high-grade glioma (HGG) remains dismal and the extent of resection correlates with overall survival and progression free disease. Epidermal growth factor receptor (EGFR) is a biomarker heterogeneously expressed in HGG. We assessed the feasibility of detecting HGG using near-infrared fluorescent antibody targeting EGFR. Mice bearing orthotopic HGG xenografts with modest EGFR expression were imaged in vivo after systemic panitumumab-IRDye800 injection to assess its tumor-specific uptake macroscopically over 14 days, and microscopically ex vivo. EGFR immunohistochemical staining of 59 tumor specimens from 35 HGG patients was scored by pathologists and expression levels were compared to that of mouse xenografts. Intratumoral distribution of panitumumab-IRDye800 correlated with near-infrared fluorescence and EGFR expression. Fluorescence distinguished tumor cells with 90% specificity and 82.5% sensitivity. Target-to-background ratios peaked at 14 h post panitumumab-IRDye800 infusion, reaching 19.5 in vivo and 7.6 ex vivo, respectively. Equivalent or higher EGFR protein expression compared to the mouse xenografts was present in 77.1% HGG patients. Age, combined with IDH-wildtype cerebral tumor, was predictive of greater EGFR protein expression in human tumors. Tumor specific uptake of panitumumab-IRDye800 provided remarkable contrast and a flexible imaging window for fluorescence-guided identification of HGGs despite modest EGFR expression.

Engineering of a novel subnanomolar affinity fibronectin III domain binder targeting human programmed death-ligand 1.

The programmed death-ligand 1 (PD-L1) is a major checkpoint protein that helps cancer cells evade the immune system. A non-invasive imaging agent with rapid clearance rate would be an ideal tool to predict and monitor the efficacy of anti-PD-L1 therapy. The aim of this research was to engineer a subnanomolar, high-affinity fibronectin type 3 domain (FN3)-based small binder targeted against human PD-L1 (hPD-L1) present on tumor cells. A naive yeast G4 library containing the FN3 gene with three binding loop sequences was used to isolate high-affinity binders targeted to purified full-length hPD-L1. The selected binder clones displayed several mutations in the loop regions of the FN3 domain. One unique clone (FN3hPD-L1-01) with a 6x His-tag at the C-terminus had a protein yield of >5 mg/L and a protein mass of 12 kDa. In vitro binding assays on six different human cancer cell lines (MDA-MB-231, DLD1, U87, 293 T, Raji and Jurkat) and murine CT26 colon carcinoma cells stably expressing hPD-L1 showed that CT26/hPD-L1 cells had the highest expression of hPD-L1 in both basal and IFN-γ-induced states, with a binding affinity of 2.38 ± 0.26 nM for FN3hPD-L1-01. The binding ability of FN3hPD-L1-01 was further confirmed by immunofluorescence staining on ex vivo CT26/hPD-L1 tumors sections. The FN3hPD-L1-01 binder represents a novel, small, high-affinity binder for imaging hPD-L1 expression on tumor cells and would aid in earlier imaging of tumors. Future clinical validation studies of the labeled FN3hPD-L1 binder(s) have the potential to monitor immune checkpoint inhibitors therapy and predict responders.

Ferumoxytol-based Dual-modality Imaging Probe for Detection of Stem Cell Transplant Rejection.

Purpose: Stem cell transplants are an effective approach to repair large bone defects. However, comprehensive techniques to monitor the fate of transplanted stem cells in vivo are lacking. Such strategies would enable corrective interventions at an early stage and greatly benefit the development of more successful tissue regeneration approaches. In this study, we designed and synthesized a dual-modality imaging probe (Feru-AFC) that can simultaneously localize transplanted stem cells and diagnose immune rejection-induced apoptosis at an early stage in vivo. Methods: We used a customized caspase-3 cleavable peptide-dye conjugate to modify the surface of clinically approved ferumoxytol nanoparticles (NPs) to generate the dual-modality imaging probe with fluorescence "light-up" feature. We labeled both mouse mesenchymal stem cells (mMSCs, matched) and pig mesenchymal stem cells (pMSCs, mismatched) with the probe and transplanted the labeled cells with biocompatible scaffold at the calvarial defects in mice. We then employed intravital microscopy (IVM) and magnetic resonance imaging (MRI) to investigate the localization, engraftment, and viability of matched and mismatched stem cells, followed by histological analyses to evaluate the results obtained from in vivo studies. Results: The Feru-AFC NPs showed good cellular uptake efficiency in the presence of lipofectin without cytotoxicity to mMSCs and pMSCs. The fluorescence of Feru-AFC NPs was turned on inside apoptotic cells due to the cleavage of peptide by activated caspase-3 and subsequent release of fluorescence dye molecules. Upon transplantation at the calvarial defects in mice, the intense fluorescence from the cleaved Feru-AFC NPs in apoptotic pMSCs was observed with a concomitant decrease in the overall cell number from days 1 to 6. In contrast, the Feru-AFC NP-treated mMSCs exhibited minimum fluorescence and the cell number also remained similar. Furthermore, in vivo MRI of the Feru-AFC NP-treated mMSC and pMSCs transplants could clearly indicate the localization of matched and mismatched cells, respectively. Conclusions: We successfully developed a dual-modality imaging probe for evaluation of the localization and viability of transplanted stem cells in mouse calvarial defects. Using ferumoxytol NPs as the platform, our Feru-AFC NPs are superparamagnetic and display a fluorescence "light-up" signature upon exposure to activated caspase-3. The results show that the probe is a promising tool for long-term stem cell tracking through MRI and early diagnosis of immune rejection-induced apoptosis through longitudinal fluorescence imaging.

Multimodal assessment of SERS nanoparticle biodistribution post ingestion reveals new potential for clinical translation of Raman imaging.

Despite extensive research and development, new nano-based diagnostic contrast agents have faced major barriers in gaining regulatory approval due to their potential systemic toxicity and prolonged retention in vital organs. Here we use five independent biodistribution techniques to demonstrate that oral ingestion of one such agent, gold-silica Raman nanoparticles, results in complete clearance with no systemic toxicity in living mice. The oral delivery mimics topical administration to the oral cavity and gastrointestinal (GI) tract as an alternative to intravenous injection. Biodistribution and clearance profiles of orally (OR) vs. intravenously (IV) administered Raman nanoparticles were assayed over the course of 48 h. Mice given either an IV or oral dose of Raman nanoparticles radiolabeled with approximately 100 µCi (3.7MBq) of 64Cu were imaged with dynamic microPET immediately post nanoparticle administration. Static microPET images were also acquired at 2 h, 5 h, 24 h and 48 h. Mice were sacrificed post imaging and various analyses were performed on the excised organs to determine nanoparticle localization. The results from microPET imaging, gamma counting, Raman imaging, ICP-MS, and hyperspectral imaging of tissue sections all correlated to reveal no evidence of systemic distribution of Raman nanoparticles after oral administration and complete clearance from the GI tract within 24 h. Paired with the unique signals and multiplexing potential of Raman nanoparticles, this approach holds great promise for realizing targeted imaging of tumors and dysplastic tissues within the oral cavity and GI-tract. Moreover, these results suggest a viable path for the first translation of high-sensitivity Raman contrast imaging into clinical practice.

A PET Imaging Strategy to Visualize Activated T Cells in Acute Graft-versus-Host Disease Elicited by Allogenic Hematopoietic Cell Transplant.

A major barrier to successful use of allogeneic hematopoietic cell transplantation is acute graft-versus-host disease (aGVHD), a devastating condition that arises when donor T cells attack host tissues. With current technologies, aGVHD diagnosis is typically made after end-organ injury and often requires invasive tests and tissue biopsies. This affects patient prognosis as treatments are dramatically less effective at late disease stages. Here, we show that a novel PET radiotracer, 2'-deoxy-2'-[18F]fluoro-9-ß-D-arabinofuranosylguanine ([18F]F-AraG), targeted toward two salvage kinase pathways preferentially accumulates in activated primary T cells. [18F]F-AraG PET imaging of a murine aGVHD model enabled visualization of secondary lymphoid organs harboring activated donor T cells prior to clinical symptoms. Tracer biodistribution in healthy humans showed favorable kinetics. This new PET strategy has great potential for early aGVHD diagnosis, enabling timely treatments and improved patient outcomes. [18F]F-AraG may be useful for imaging activated T cells in various biomedical applications. Cancer Res; 77(11); 2893-902. ©2017 AACR.

Optical imaging with her2-targeted affibody molecules can monitor hsp90 treatment response in a breast cancer xenograft mouse model.

PURPOSE: To determine whether optical imaging can be used for in vivo therapy response monitoring as an alternative to radionuclide techniques. For this, we evaluated the known Her2 response to 17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride (17-DMAG) treatment, an Hsp90 inhibitor. EXPERIMENTAL DESIGN: After in vitro 17-DMAG treatment response evaluation of MCF7 parental cells and 2 HER2-transfected clones (clone A medium, B high Her2 expression), we established human breast cancer xenografts in nude mice (only parental and clone B) for in vivo evaluation. Mice received 120 mg/kg of 17-DMAG in 4 doses at 12-hour intervals intraperitonially (n = 14) or PBS as carrier control (n = 9). Optical images were obtained both pretreatment (day 0) and posttreatment (day 3, 6, and 9), always 5 hours postinjection of 500 pmol of anti-Her2 Affibody-AlexaFluor680 via tail vein (with preinjection background subtraction). Days 3 and 9 in vivo optical imaging signal was further correlated with ex vivo Her2 levels by Western blot after sacrifice. RESULTS: Her2 expression decreased with 17-DMAG dose in vitro. In vivo optical imaging signal was reduced by 22.5% in clone B (P = 0.003) and by 9% in MCF7 parental tumors (P = 0.23) 3 days after 17-DMAG treatment; optical imaging signal recovered in both tumor types at days 6 to 9. In the carrier group, no signal reduction was observed. Pearson correlation of in vivo optical imaging signal with ex vivo Her2 levels ranged from 0.73 to 0.89. CONCLUSIONS: Optical imaging with an affibody can be used to noninvasively monitor changes in Her2 expression in vivo as a response to treatment with an Hsp90 inhibitor, with results similar to response measurements in positron emission tomography imaging studies.

Molecular imaging of the efficacy of heat shock protein 90 inhibitors in living subjects.

Heat shock protein 90 alpha (Hsp90 alpha)/p23 and Hsp90 beta/p23 interactions are crucial for proper folding of proteins involved in cancer and neurodegenerative diseases. Small molecule Hsp90 inhibitors block Hsp90 alpha/p23 and Hsp90 beta/p23 interactions in part by preventing ATP binding to Hsp90. The importance of isoform-selective Hsp90 alpha/p23 and Hsp90 beta/p23 interactions in determining the sensitivity to Hsp90 was examined using 293T human kidney cancer cells stably expressing split Renilla luciferase (RL) reporters. Interactions between Hsp90 alpha/p23 and Hsp90 beta/p23 in the split RL reporters led to complementation of RL activity, which was determined by bioluminescence imaging of intact cells in cell culture and living mice using a cooled charge-coupled device camera. The three geldanamycin-based and seven purine-scaffold Hsp90 inhibitors led to different levels of inhibition of complemented RL activities (10-70%). However, there was no isoform selectivity to both classes of Hsp90 inhibitors in cell culture conditions. The most potent Hsp90 inhibitor, PU-H71, however, led to a 60% and 30% decrease in RL activity (14 hr) in 293T xenografts expressing Hsp90 alpha/p23 and Hsp90 beta/p23 split reporters respectively, relative to carrier control-treated mice. Molecular imaging of isoform-specific Hsp90 alpha/p23 and Hsp90 beta/p23 interactions and efficacy of different classes of Hsp90 inhibitors in living subjects have been achieved with a novel genetically encoded reporter gene strategy that should help in accelerating development of potent and isoform-selective Hsp90 inhibitors.

Fluorescent fructose derivatives for imaging breast cancer cells.

Breast cancer cells are known to overexpress Glut5, a sugar transporter responsible for the transfer of fructose across the cell membrane. Since Glut5 transporter is not significantly expressed in normal breast cells, fructose uptake can potentially be used to differentiate between normal and cancerous cells. Fructose was labeled with two fluorophores at the C-1 position: 7-nitro-1,2,3-benzadiazole (NBD) and Cy5.5. The labeling site was chosen on the basis of the presence and substrate specificity of the key proteins involved in the first steps of fructose metabolism. Using fluorescence microscopy, the uptake of the probes was studied in three breast cancer cell lines: MCF 7, MDA-MB-435, and MDA-MB-231. Both fluorescent fructose derivatives showed a very good uptake in all tested cell lines. The level of uptake was comparable to that of the corresponding glucose analogs, 2-NBDG and Cy5.5-DG. Significant uptake of 1-NBDF derivative was not observed in cells lacking Glut5 transporter, while the uptake of the 1-Cy5.5-DF derivative was independent of the presence of a fructose-specific transporter. While 1-NBDF showed Glut5-specific accumulation, the coupling of a large fluorophore such as Cy5.5 likely introduces big structural and electronic changes, leading to a fructose derivative that does not accurately describe the uptake of fructose in cells.

Split luciferase complementation assay for studying interaction of proteins x and y in living mice.

This protocol describes a split luciferase complementation assay that can be used to repetitively and noninvasively study the interaction of proteins in small living animals. After the expression of the appropriate vectors has been checked in cell culture in vivo, studies can be performed either by implanting transiently transfected cells for short-term analysis (maximum of 7 days), as described below, or with tumor models grown from tumor cells stably expressing the complete reporter system. For optical imaging, the number of cells implanted can be relatively low (~1-5 × 10(6)), and imaging can begin even before the tumors are palpable. When using a regulated reporter gene, it may be necessary to perform a dose-dependent pilot experiment with the inducer or repressor before performing the primary experiments. Animal models other than mice can be used, including rats and, in theory, transgenic animals in which the reporter constructs have been stably integrated into the genome. Animals larger than the rat would be difficult to image due to poor penetration of light. For these larger-animal models, the use of other imaging technologies such as microPET should be considered.

Split luciferase complementation assay for studying interaction of proteins x and y in cells.

This protocol describes a split luciferase complementation assay used to study the interaction of proteins in cells. In the "split protein" strategy, a single reporter protein/enzyme (in this case, firefly luciferase [Fluc]) is cleaved into amino-terminal and carboxy-terminal halves; each half is fused to one of two interacting proteins, X and Y. Physical interactions between the two proteins, X and Y, reconstitute the functional reporter protein, leading to enzymatic activities that can be measured by in vitro or in vivo assays. It is important to perform the assay initially in cell culture before proceeding with animal imaging, not only to conserve animals, but also to establish the sensitivity of the assay.