Purification of proteins from a heterogeneous mixture often involves a multi-step process that makes use of the physical, chemical, and electrical properties of the protein being purified. Important properties of a protein that are relevant to its purification are (a) solubility, which determines the ability of the protein to remain in solution or to precipitate out in the presence of salt; (b) charge, which is an important property relevant to ion exchange chromatography and isoelectric focusing; (c) size, which is relevant in processes involving dialysis, gel-filtration chromatography, gel electrophoresis and sedimentation velocity; (d) specific binding, which allows purification of a protein based on its binding to a ligand; and (e) ability to form complexes in the presence of other reagents, such as in antibody precipitation. Protein detection and purification has become a major focus of research activities in view of the challenges faced by researchers involved in functional genomics and proteomics.
Tetanus toxin fragment C (TTC) is a 50 kD non-toxic polypeptide that is one of the products of cleavage of tetanus toxin by papain. Previous studies indicates that TTC in all its forms is highly insoluble and difficult to purify without resorting to denaturing condition. Denaturing conditions include the use of 6M Guanidine Chloride or 6-8 M Urea for solubilization of protein inclusion bodies post bacterial pellet suspension in 20 mM Tris-HCL (pH 8) and lysation with a French Press. Protein purification under denaturing conditions unfolds TTC and linearizes the 3-dimensional structure needed for biological activity. Protein refolding from this linearized form is difficult, but can be accomplished by means of a multistep dialysis with a gradual decrease in amount of denaturing agent. The refolding process is complex and not always successful.
Nerve function may be evaluated using electrophysiology/electromyography (EMG) EMG is painful and invasive; most patients do not tolerate it well. EMG is limited in what nerves it can evaluate, and can for example, not evaluate the spinal cord's function itself directly because of the need for stimulating and sensing needles to be inserted proximally and distally into the neuromuscular or neurosensor units being investigated.
The present disclosure, according to specific example embodiments, generally relates to protein purification and imaging. In particular, the present disclosure relates to a Tetanus Toxin Fragment C (TTC) based imaging agent and associated methods of use, as well as methods to process confocal microscopy datasets. The TTC based imaging agents of the present disclosure generally comprise a Tetanus Toxin Fragment C and a reporter, and such imaging agents may be useful diagnostically, for example, as a means of investigating nerve diseases of various types.
The present disclosure, according to specific example embodiments, also provides methods comprising processing confocal microscopy datasets to provide a 360 degree average fluorescence intensity profile from the center of a spheroid towards the outer edge of the spheroid. Such methods, among other things, allows for quantitative characterization of spatial heterogeneity and temporal dynamics of fluorescence distribution within multi-cellular 3D spheroids.
Some specific example embodiments of the disclosure may be understood by referring, in part, to the following description and the accompanying drawings.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
While the present disclosure is susceptible to various modifications and alternative forms, specific example embodiments have been shown in the drawings and are described in more detail below. It should be understood, however, that the description of specific example embodiments is not intended to limit the invention to the particular forms disclosed, but on the contrary, this disclosure is to cover all modifications and equivalents as illustrated, in part, by the appended claims.
The present disclosure, according to certain embodiments, provides methods for purifying TTC comprising obtaining a supernatant comprising soluble TTC and purifying TTC from the supernatant under native conditions to obtain a substantially purified TTC. Such methods may avoid denaturation of TTC, and thus may preserve the biologically active conformation of TTC. In certain embodiments, the TTC may be His-tagged, and such His-tagged TTC may be purified using a column based purification kit, for example, nickel coated sephadex beads and imidazole.
The present disclosure, according to certain embodiments, provides imaging agents comprising TTC and a reporter. Such imaging agents may allow imaging the process of retrograde axonal transport, among other things. The TTC in the imaging agent may be the complete TTC protein or fragment thereof, so long as it retains biological activity. In this context, biological activity may refer to the properties of neuronal uptake and retrograde transport, which TTC possesses. The TTC is associated with a reporter to allow the detection of TTC activity (e.g., neuronal uptake and retrograde transport). The reporter may be any molecule that produces signal detectable by various non-invasive and invasive imaging technologies. Examples of reporters include fluorescent labels and radiolabels such as, for example, Alexa fluors, fluorescent dyes, green fluorescent proteins, red fluorescent proteins, Alexa dyes, and indium. Imaging technologies that may be used in conjunction with the imaging agents of the present disclosure, include, but are not limited to, magnetic resonance imaging (MRI), positron emission tomography (PET), and computed tomography (CT). In certain embodiments, the imaging agent of the present disclosure may be adapted to carry not only a reporter, but instead or in addition, a therapeutic moiety such as a drug, growth factor, radiation emitting compound or the like, allowing the compound to be used for therapeutic purposes in addition to, or instead of diagnostic applications. Accordingly, imaging agents of the present disclosure may be used in in methods for imaging retrograde axonl transport and methods to detect and/or treat a variety of peripheral nerve diseases. In these methods, the imaging agent may be injected into a mammal and a signal may be detected.
The present disclosure also provides, according to certain embodiments, a methods for processing confocal microscopy datasets to provide a 360 degree average fluorescence intensity profile from the center of spheroid towards the outer edge of the spheroid. As used herein, the term “spheroids” refers to three-dimensional aggregates of cells that serve as in vitro models of tumors, and model cancerous processes more closely than do monolayer cultures of cancer cells. In certain embodiment, spheroid refers to other cells, tissues, or cell-tissue constructs of biological relevance could be studied with similar strategies incorporating fluorescent reporters and suitable promoters in conjunction with the methods of the present disclosure. In certain embodiments, the cells of interest may be a portion of a tumor spheroid. In certain other embodiments, any compound comprising a reporter may be studied using the methods to process confocol microscopy datasets.
In one embodiment, an average radial profile image analysis on a user specified central image slice through the spheroid may be performed. The RFP channel may be used to threshold the data and to determine the center of the spheroid. Using this computer determined center as a fulcrum, a radial arc was swept through user specified 360 degrees, while plotting an expression plot profile along each radius (plot line thickness=1 pixel) from a reporter (e.g., a fluorescent reporter). Such methods may be used to analyze the large image datasets of spheroids and automatically determine the center, radius, and radial intensity profile of a spheroid. Profiles generated as a result of various experimental conditions may be analyzed with this method in this manner with minimal user interaction. The flow chart (
In certain embodiments, the methods of the present disclosure may be a macro in software. In certain other embodiments, the methods of the present disclosure may be implemented as a separate image analysis program, or as a component of a larger image analysis software platform.
One example of a method of the processing confocal microscopy datasets may be executed in the form of a macro. For example, the text of a working macro that works with v1.35s of the ImageJ program as obtained from http://rsb.info.nih.gov/ij/ if provided below. This macro serves to demonstrate a working implementation of one example for processing confocal microscopy datasets:
In one specific embodiment, the profiles of a spheroid comprised of cells expressing both Red Fluorescent Protein (RFP) under control of a constitutive CMV promoter and Green Fluorescent Protein (GFP) under control of a dxBE (Hypoxic Responsive Element) promoter are compared and have utility as a model of hypoxia in tumor cells. For example, an algorithm may be used for the analysis of biochemical events (in this case hypoxia as a function of distance from the center of the spheroid) in 3D space in a quantitative semi-automatic manner. The methods of the present disclosure allow analysis of these complex data.
Therefore, the present invention is well adapted to attain the ends and advantages mentioned as well as those that are inherent therein. While numerous changes may be made by those skilled in the art, such changes are encompassed within the spirit of this invention as illustrated, in part, by the appended claims.
Purification of TTC
Three bacterial pellets were combined and induced with 1 mMIPTG at OD 0.6 at 30° C. The pellets were solubilized with 0.1 mg/mL Lysozyme in 20 mM Tris-HCL+500 mM NaCl. Pellets were stirred for 1 hour at room temperature and this fraction was analyzed for solubilized TTC in native conditions. The fraction was sonicated 30 sec (3 times) with 60 sec breaks and then Spun at 8000 g for 20 minutes (clear post lysis supernatant+pellet). The supernatant and the small pellet were analyzed after denaturing conditions Denaturing conditions refers to exposing the inclusion body pellet to Urea for 3 hours, and spun down at 8000 g for 20 min, purify using standard methods with His-Nickel coated beads. Native conditions refer to natively collected supernatant fraction purified using standard methods with His-Nickel coated beads.
As shown in
TTC Fluorescent Labeling
To label TTC and demonstrate retention of biological activity of the compound, an Alexa fluor 680 protein labeling kit was used (Molecular probes-A20172). Purified TTC was labeled with initial concentration of 2 mg/ml (500 ul). 50 ul of 1M NaCO3 buffer to TTC. The total fraction of TTC (550 ul) was placed over column. Collection light blue band, 30 min after application. 3 fractions were collected and analyzed (
Agent to Image Retrograde Axonal Transport
The TTC plasmid DH5 alpha competent cells were subcloned and the sequenced DNA was similar to the published sequence. Protein expression and purification was performed in Epicurian Coli BL31 DE3 using standard methods. The purity and integrity of the protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The immunoreactivity of the TTC protein was confirmed via Western blotting and ELISA assays using a mouse monoclonal antibody to the C-fragment of tetanus toxin (Roche #11 131 621 001). The integrity and immunoreactivity of the Tetanus toxin C protein and the derivatives we have prepared remained constant. Cell uptake assays were performed in cultured PC12 cells with Alexa488 and Alexa688 labeled TTC and the positive results from these studies confirmed the structural and functional integrity of the recombinant protein, post purification. Optically and nuclear labeled compound were injected into the soleus muscles of C57bl mice, and performed CT-SPECT imaging studies and biodistribution studies, which indicated nerve uptake of the intramuscularly injected compound. In vivo optical imaging of the sciatic nerve was performed with the Xenogen IVIS 200 fluorescent imager and with the Mauna Kea Cell-vizio fiberoptic system, and also demonstrated nerve uptake of the compound after intramuscular injection. The whole body pharmacokinetics of the labeled nuclear compound has been measured, and found it to be modeled by a biexponential fit with t1/2alpha:=1.115 h (75.3% contribution) and t1/2beta=95.738 h (24.7%) after intramuscular injection into the soleus muscle Cell Studies with Alexa-TTC
PC-12 cells (ATCC, CRL-1721), pheochromocytome cells from rat adrenal gland were cultured in DMfEM/F12 with 15% horse serum. Cells were grown on slides coated with 10% matrigel for 24 hours to 20% confluence. The cells were differentiated with 15 ng/ml NGF overnight. The cells were incubated with 4 ug Alexa-TTC/250 μl media for 4 hours. The cells were viewed using confocal microscopy, Olympus FluoviewFV 1000 (
TTC Uptake and Transport
3 C57BL6 mice were injected with 80 ug/20 uL TTC-Alexa488 in the right soleus and 40 ug/20 uL HAS-Alexa680 in the left soleus and were sacrificed after 5, 24, and 36 hours. During the time between injection and sacrifice, as well as after sacrifice, one or more images of each mouse were taken with an OV100 fluorescent imager (
The Effect of Temperature Changes and DOTA Chelation on the Immunoreactivity of His-tagged TTC
His-tagged TTC was stored during a 24-hour period under varying temperature conditions including: 4 degrees Celsius, room temperature (27 degrees Celsius), 37 degrees Celsius, 43 degrees Celsius, and combinations thereof Following the 24-hour period, the proteins were run on an SDS-PAGE gel, followed by Western blotting and immunodetection. An ELISA was also performed on the samples. This experiment was performed on two occasions, the first shown in
Neuronal Labeling and Immunodetection of His-TTC in PC 12 Cells
PC-12 cells were seeded at a density of 20 000 cells/well and exposed to NGF on 12 mm glass coverslips covered with poly-D-lysine (Sigma). The cells were then left to attach and form neural processes for 2.5 days. Cells began forming neural outgrowths and were at about 30% confluency when grown on poly-D-lysine coverslips. Cells on clear uncoated coverslips were attached poorly and had less neural processes. Cells on coverslips were then removed from media and excess fluid removed by Kimwipes. The cells were subsequently exposed to TTC in 0.1M Na2PO4 buffer (pH 8.5) labeled with NHS-DOTA at 4° C. or 25° C. with either 1:100 or 1:200 excess DOTA. All protein was solubilized in 20 uL droplets of PBS and PC12 cells on Coverslips were exposed to these droplets, covering all cells for 85 minutes at 37° C. in a humid cell culture incubator. After incubation, cells were washed and then fixed with 5% formalin for 5 minutes. Post-fixation, cells were washed and then exposed to an antibody regimen consisting of exposure to a primary antibody at 5 mg/ml (TC Roche Cat #1 131 621 batch 933 53220) for 1 hour followed by 3 washes and subsequent exposure to a secondary antibody 1:100 (2.5 uL: 250 uL) Zymed anti FitC (Cat# 81 65511 batch 505 94880) for 30 minutes followed by 3 washes. The cells were then mounted in Molecular Probes anti-fading medium and viewed with a Confocal FV 1000 microscope.
Animal Imaging
200 ug of Alexa680-TTC was injected into the gastrocnemicus muscle in 200 uL of PBS. Imaging was performed on the XenogenIVIS 200 system using the CY5.5 filter set through various phases of dissection at 24 hours after the injection (
Alexa680-TTC In Vivo Assay
The in vivo distribution of TTC was evaluated using the Ivis200 imager over a period of 12 hours. The mouse was C57BL/6. In this in vivo time course study, Alexa680-TTC was injected into the gastrocnemicus (50 ug/50 uL) in C57BL/6 mice (
TTC-His Conjugation with EC
0.15 mg ethylenedicysteine (EC), 0.12 mg N-hydroxysulfosuccinimide (Sulfo-NHS), and 0.107 mg 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were added to 1 mL of 1.5 mg/ml TTC-His. Sulfo-NHS and EDC are the catalysts for the conjugation. The mixture was permitted to react overnight at room temperature. The protein was then dialysed (MW<10.000) for 8 hours, changing the dialysate every hour. The product was then freeze dried. Following the conjugation procedure, the sample underwent SDS-PAGE, Western blotting and immunodetection, and an ELISA assay with appropriate controls, as shown in
PC12 Uptake Studies
Uptake studies on PC12 cells were performed with the various TC conjugates, PC12 cells were seeded in 96-well flat-bottom plates at a density of 2,000 cells/well. After 24 hours, 50 ng/ml NGF was added to the media. Media was then changed at 2 and 5 days after seeding, and the uptake study was performed 8 days after seeding.
The uptake study was a multi-step process. First, 1-3 ug TC-Alexa488, 1-3 ug HSA-Alexa 860 and combinations of both were added to cells. Uptake of the conjugates was observed under a confocal microscope at 37° C. for 1 hour. A second step involved repeating the above step, followed by fixation of the cells after 1 hour with 5% paraformaldehyde at room temperature for 5 min. Cells were then washed and observed under a confocal microscope. A positive control (TC-Roche) was used in this experiment. The cells were first exposed to a primary anti-TC monoclonal antibody (diluted 1:2000) for 1 hour and then to a secondary anti-FITC anti-body (diluted 1:2000) for 30 minutes. Following two washes with 0.5% BSA in PBS, the cells were observed with an FV1000 confocal microscope (
TTC-DOTA-Indium Labeling and Conjugating TTC to NHS-DOTA
TTC was dialyzed overnight to 1 L Tris 0.3 M, pH=8, with Chelex 100 1.2 g. The TTC was incubated with NHS-DOTA at molar excess of 20, 100, and 200 at 25 ° C. for 24 h with end over end mixing. The protein was dialyzed again to 1 L Tris 0.3 M, pH=8 and Chelex. Indium-trichloride was prepared with ammonium acetate and citric acid to a weak citrate-acetate chelate. This weakly chelated Indium was incubated with TTC-DOTA which then transchelates the Indium to DOTA. Immunoreactivity of the conjugate (A37) was assessed from ELISA assay (
For the number of chelex per protein molecules, have an iTLCassay is still needed. The results indicate that 1 ug of conjugate gives % of immunoreaction (% of control) at around 98%, although the OD value of 1 ug TTC showed that it is out of scale. The 0.25 ug and the 0.125 ug gives close to consisitant results. (
Indium-111 Labeling of TTC-DOTA
600 uL of 0.3 M ammonium acetate at pH 9 was mixed with 400 uL In-111-trichloride in 0.05 HCl at pH 1-1.4. After 10-15 minutes, 250 uL of “In-Acetate” solution was transpipetted to each of 4 protein-DOTA conjugates. DOTA20, DOTA100, DOTA200A and DOTA200B. The samples were allowed to incubate overnight at room temperature. Table 1 below shows the TTC-DOTA-Indium labeling. This indicated that very poor labeling was achieved. Heating at 43° C. for 1 hour did not improve the results.
Thin layer chromatography (TLC) was performed on the samples. 80:20 MetOH:Water on Cellulose does not appear to separate ionic Indium-T111 and Indium-Acetate. TLC cannot be used to assess labeling in its present form (
Optimization of Indium-Acetate (Citrate) weakly chelated species in solution was assessed using TLC with respect to pH and time. (
Animal Studies
MCAM imaging procedure and coded aperture was used as shown in
Development of a Nerve Tracking Compound (NTC) and Nuclear and Optical Imaging Study
The base protein (TTC) was purified, and labeled with NHS-DOTA-111Indium for nuclear imaging studies and with NHS-Alexa488 or NHS-Alexa688 for optical imaging studies. NTC was injected into the soleus muscle of C57bl mice, and nuclear SPECT-CT imaging performed with the GammaMedica Xspect device, optical in vivo imaging was performed with the Mauna Kea Cell-Vizio LSU-488 system using a S-300-5.0 Proflex fiberoptic probe and the Xenogen IVIS 200 Fluorescent imager, while ex vivo microscopy was performed with the Olympus laser scanning confocal microscope and with an epifluorescence microscope. Bio-distribution studies and histological studies were undertaken. The studies indicated that NTC was taken up in the sciatic nerve after intramuscular injection into the soleus muscle. SPECT-CT images showed distribution along the nerve, confirmed by bio-distribution studies, which demonstrated 6.97±4.6% ID/g (mean±SD) in the ipsilateral sciatic, which was 363 fold higher than the contralateral non-injected side at 24 hours after injection. In vivo optical imaging demonstrated uptake in the sciatic nerve, while histological studies of excised nerve segments confirmed uptake in nerve fassicles within the sciatic nerve. Pharmacokinetic 2-compartment modeling yielded t1/2alpha=1.1 h and t1/2 beta=95.7 h (75.3% and 24.7% contribution respectively). Therefore, labeled NTC is taken up into motor nerve endings after intramuscular injection, and is retrogradely transported in axons. This process is traceable using multiple imaging technologies, and may be useful in the evaluation and treatment of nerve diseases.
Real Time Examination of Alexa488-TTC Sciatic Nerve Distribution
C75BL6 mice were injected with 15 uL or 50 uL of 1.5 mg/ml Alexa488-TTC in the gastrocnemius. The mice were anesthetized with isofluorane at various time points, ranging from 15 minutes to 4.25 hours, and the sciatic nerves were opened for imaging, as shown in
Molecular Imaging of Tumor Spheroids for Screening of Novel Inhibitors of HIF1alpha Signaling.
Hypoxia plays a major role in tumor progression, tumor angiogenesis, and resistance to chemo- and radiotherapy. Hypoxia inducible factor-1α (HIF-1α) is an important regulator of the molecular signaling mechanisms involved in the response to hypoxia. Drugs capable of blocking HIF-1α may be very efficient for anticancer therapy. The goal of this investigation was to assess which of the novel drugs with different mechanisms of action may inhibit or potentiate the inhibition of HIF-1α expression and activity in tumor cell spheroids under hypoxia.
The image analysis software developed in this study would provide 360° average fluorescence intensity profile from the center of spheroid towards the outer edge of the spheroid. This digital tool was used to analyze 3D multi-cellular spheroids of tumor cells bearing HIF-1α-specific dual fluorescence protein reporter system.
The C6#4 reporter cell line constitutively expresses DsRed2/XPRT reporter fusion protein and HIF1α-inducible HSV1tk/GFP fusion reporter protein. Hypoxic core in spheroids of C6#4 cells developed after spheroids grew to more than 350 um in size, as visualized by dynamic quantitative confocal fluorescence microscopy system FV1000 (Olympus) (
Spheroids grow larger over time; their centers gradually become hypoxic, as indicated by the induction of the HIF1-alpha pathway visualized by the expression of GFP. Subjecting spheroids to hypoxic experimental conditions (Cobalt chloride) rapidly induces hypoxia in the entire spheroid within 6-8 hours, while untreated spheroids developed hypoxic cores after about 3 days in culture. This hypoxic response is inhibited by a Hif 1-alpha inhibitor, PX 478. Cellular motility is affected by hypoxia, and is currently under study. Prior to the methods of the present disclosure, the analysis of the spheroids were being done based on the overall intensity values and manually extracting radial profiles. In practice, this is prohibitively expensive of labor and not feasible to complete for a large numbers of spheroids.
Quantitation of Spatial and Temporal Dynamics of Expression of Fluorescent Reporter Proteins in Multi-Cellular Tumor Spheroids
Custom software was written to perform average radial profile image analysis on the user specified central image slice through the spheroid. The RFP channel was used to threshold the data and to determine the center of the spheroid. Using this computer determined center as a fulcrum, a radial arc was swept through user specified 360 degrees, while plotting a GFP and RFP expression plot profile along each radius (plot line thickness=1 pixel). Microscopy imaging datasets (Olympus FV-1000) included constitutively expressed RFP and HIF-1α-inducible GFP channels acquired at 20 μm intervals using a 800×800 imaging matrix/image for a typical imaging stack of 12 images/spheroid over 5-7 days. Image datasets were analyzed with the new software and displayed as GFP/RFP intensity ratio as a function over a distance along the maximum radius. Spheroids of 710±20 um in diameter developed within 3 days a “ring-shaped” hypoxic area with a peak of HIF-1α-induced GFP fluorescence at 120±30 um from the spheroid center. Over the following 3 days, this hypoxic ring gradually extended towards spheroid periphery, with stellar-like extensions towards spheroid periphery and increased fluorescence intensity, reflecting pathways of hypoxic cell migration. Spheroid border was populated with several layers of highly GFP-positive cells with persistent HIF-1α signaling activity. The newly developed software tool for measurement of average radial fluorescence intensity profiles in confocal fluorescence microscopy images of 3D spheroids and allows for quantitative characterization of spatial heterogeneity and temporal dynamics of fluorescence distribution within multi-cellular 3D spheroids (
Therefore, the present invention is well adapted to attain the ends and advantages mentioned as well as those that are inherent therein. While numerous changes may be made by those skilled in the art, such changes are encompassed within the spirit of this invention as illustrated, in part, by the appended claims.
This application claims the benefit of U.S. Provisional Application Ser. No. 60/806,375 filed on Jun. 30, 2006, which is incorporated by reference.
Number | Date | Country | |
---|---|---|---|
60806375 | Jun 2006 | US |