This work was supported by the Swiss National Science Foundation (grant no. 320030-184713). Authors BBT and KC are supported by the Velux Foundation (project n. 1123). Author ST received support from the Swiss National Science Foundation (Early Post-Doc Mobility Scholarship, no. P2GEP3_191446), the Prof Dr. Max Cloetta Foundation (Clinical Medicine Plus scholarship), and the Jean and Madeleine Vachoux Foundation.

All experimental procedures were conducted in agreement with the Ethics Committee for Human and Animal Experimentation of the Canton of Geneva, the Cantonal Commission for Research Ethics (CCER), and the General Direction Of The Health Of The Canton Of Geneva (Switzerland), respectively. Data are reported following Animal Research: Reporting In-vivo Experiments (ARRIVE) guidelines.
1. SPECT camera preparation and calibration
<ol>
	<li>Turn on the camera, load the operating software (see Table of Materials). Click on the Home XYZ Stage button to perform homing.</li>
	<li>Set up the experiment composed of one 10 min scan acquisition. Set the Step mode to Fine and the Acquisition mode to the Listmode (to record the entire emission spectrum).</li>
	<li>Set up the bed and ensure that the warming system, breathing sensor, and anesthesia are functional and secure (Figure 1A-D). Then, place a phantom (i.e., 2 mL of a known concentration of 125I in a 2 mL microfuge tube) where the animal&#39;s head will be positioned (centered on the area, Figure 1E).</li>
	<li>Set the scan area by sliding the cursors for the three dimensions with the help of the three images in the bottom part of the screen. Ensure that the scan volume of the phantom and the animal is the same.</li>
	<li>Start the phantom scan for subsequent calibration with the parameters established in steps 1.2-1.4.</li>
</ol>
2. Workspace setup for SPECT imaging
<ol>
	<li>Clean the workspace with disinfectant virucide and place soft papers on all the surfaces.</li>
	<li>Ensure that there is enough isoflurane and oxygen in their respective tanks.</li>
	<li>Prepare a 24 G catheter by cutting off the fins of the butterfly catheter to have a clearer view of the rat tail vein.</li>
	<li>Coat the catheter by filling it completely with heparin solution (25000 U/mL) after removing the needle. Then, place the needle back in it to avoid blood clot formation following catheter insertion.</li>
</ol>
3. [125I]CLINDE radiotracer synthesis
CAUTION: Radioactivity can have sufficient ionizing energy to affect the atoms of the living cells and damage their genetic material (DNA).
<ol>
	<li>Ensure to work in an appropriate environment, authorized for experiments involving radioactivity. 
	NOTE: Wear the appropriate personal protective equipment (PPE), for radioactivity handling, including finger and body dosimeters. Try to stay at a safe distance from any source of radioactivity.</li>
	<li>Incubate 100 &#181;g of tributylin precursor in 100 &#181;L of acetic acid with sodium iodide (Na125I) (see Table of Materials) and 5 &#181;L of 37% peracetic acid at 70 &#176;C for 20 min in a thermocycler positioned in a glove box.</li>
	<li>Dilute the reaction using 50% acetonitrile (ACN) in water to reach a volume of 500 &#181;L. Inject the 500 &#181;L of the diluted reaction onto a reverse-phase column (see Table of Materials).</li>
	<li>Isolate the [125I]CLINDE with a linear gradient HPLC run from 5%-95% ACN in 7 mM H3PO4 for 10 min. Dilute the isolate in H2O to attain a final volume of 10 mL, and then inject the diluted reaction onto a concentration column (see Table of Materials).</li>
	<li>Elute the [125I]CLINDE from the column with 300 &#181;L of absolute ethanol, and then evaporate the ethanol by incubating it in a vacuum centrifuge at RT for 40 min.</li>
	<li>Dilute the residue containing the [125I]CLINDE in 300 &#181;L of sterile saline to create a stock solution.</li>
	<li>After measuring its radioactivity, dilute the stock solution in sterile saline to obtain a solution of 0.037 MBq in 500 &#181;L.</li>
	<li>Purify the tracer by HPLC (High-performance liquid chromatography). Determine the elution time using a standard calibration at 450 nm, and isolate the single radioactive peak. Perform the standard calibration curve using seven standard concentrations of cold (non-radioactive) CLINDE (0.1 &#181;g, 0.5 &#181;g, 0.75 &#181;g, 1 &#181;g, 1.5 &#181;g, 2 &#181;g, and 5 &#181;g in 400 &#181;L of a solution of 50% ACN). Establish the radiochemical purity by measuring a single peak in radio TLC (Thin layer chromatography). Ensure that the purity of the radiochemical is above 60%.</li>
	<li>Check the specific activity of the radioligand measuring ultraviolet absorbance at 450 nm and the calibration curves established with cold reference compounds. Ensure that the specific activity is greater than 1000 GBq/&#181;mol.</li>
</ol>
4. [125I]R91150 radiotracer synthesis
NOTE: Ensure to follow the same security rules as mentioned in the CLINDE synthesis section.
<ol>
	<li>Mix 300 &#181;g of R91150 precursor in a solution of 3 &#181;L of absolute ethanol, 3 &#181;L of glacial acetic acid, 15 &#181;L of carrier-free Na125I (see Table of Materials) (10 mCi) in 0.05 M NaOH and 3 &#181;L of 30% H2O2. Incubate for 30 min in a glove box.</li>
	<li>Inject the entire reaction into a reverse-phase column (see Table of Materials).</li>
	<li>Isolate [125I]R91150 by isocratic HPLC run (ACN/water 50/50, 10 mM acetic acid buffer) with a flow rate of 3 mL/min.</li>
	<li>Dilute [125I]R91150 in H2O for a final volume of 10 mL.</li>
	<li>Inject 10 mL of the diluted reaction onto a concentration column (see Table of Materials).</li>
	<li>Elute [125I]R91150 from the column with 300 &#181;L of absolute ethanol.</li>
	<li>Evaporate the ethanol by incubating it in a vacuum centrifuge at RT for 40 min.</li>
	<li>Dilute the residue containing [125I]R91150 in 300 &#181;L of saline.</li>
	<li>Purify the tracer by HPLC. Determine the elution time using a standard calibration at 450 nm and isolate the single radioactive peak. Establish the radiochemical purity by measuring a single peak in radio TLC. Ensure that the purity of the radiochemical is above 98%.</li>
</ol>
5. Animal preparation
<ol>
	<li>Weigh and anesthetize the TgF344-AD rat (male or female, from 2-24 months old) in an induction chamber with 3% isoflurane. Once deeply anesthetized, lower the isoflurane flow to 2% (0.4 L/min, 100% O2) in the chamber.</li>
	<li>Position the animal on a pre-warmed bed equipped with an anesthesia nosecone. Maintain isoflurane at 2% (0.4 L/min, 100% O2).</li>
	<li>Apply eye gel lubricant in the animal&#39;s eyes and confirm the depth of anesthesia via respiratory monitoring; adjust anesthesia if necessary.</li>
	<li>Perform the 24 G catheter insertion in the tail vein.</li>
</ol>
6. SPECT acquisition
<ol>
	<li>Transfer the animal to the camera bed&#160;equipped with a temperature-controlled heating pad set to 38&#176;C (Figure 1E).</li>
	<li>Secure the head of the animal onto the bite bar and fix the head supports.</li>
	<li>Set up the experiment on a 60 min scan constituting 60 frames of 1 min. Reuse all other parameters set up in step 1. Click on the Update Image button to update the animal position.</li>
	<li>Set the scan area by sliding the cursors with the help of the three images in the bottom part of the screen for the three dimensions.</li>
	<li>Inject 500 &#181;L of the radioactive radiotracer ([125I]CLINDE or [125I]R91150), and then flush the tube with 300 &#181;L of sterile 0.9% NaCl. Simultaneously click on Start Acquisition to start the scan.</li>
	<li>During the scan time, ensure to maintain the animal under constant anesthesia with respiration rate monitoring. Adjust the flow of isoflurane if needed.</li>
	<li>Once the scan is over, quickly euthanize the anesthetized animal by decapitation.</li>
</ol>
7. Scan reconstruction
<ol>
	<li>Open the scan reconstruction software (see Table of Materials), and then open the data set, looking for the [filename].parameters file, created in the folder of the scan.</li>
	<li>Select the isotope of interest. Note that the Listmode parameter allows multiple isotope selection at this step.</li>
	<li>Select the following parameters: 0.4 mm Voxel size, 4 (POS-EM) subsets, 6 iterations (24ME-EM equivalent), no post-filter, and the isotope corresponding Decay Correction. Select the output format to NIfTI, and then select Start SPECT reconstruction.</li>
</ol>
8. Rat brain extraction
<ol>
	<li>In the same anesthetic event as the scanning procedure&#160;and after ensuring the deep anesthesia state of the animal by&#160;monitoring&#160;its respiratory rate, proceed to the decapitation by guillotine and quickly transfer the head to the dissection bench.</li>
	<li>With scissors, carefully cut the skin on top of the head from the back to the front till the middle of the eyes.</li>
	<li>Cut off the excess muscles around the base of the skull and the cervical vertebras.</li>
	<li>Next, carefully place one blade of the scissors in the hole at the back of the skull, the foramen magnum, and remove the back part of the skull with surgical pliers.</li>
	<li>Then, with surgical pliers, carefully remove the top part of the skull. The skull of old male rats can be thick, remove as small pieces to avoid damage to the brain.</li>
	<li>Carefully cut the meninges with scissors. Meninges can damage the brain during the extraction process; remove it as a precaution.</li>
	<li>After removing the top part of the skull, turn the head of the animal around, and with a small flat spatula, carefully pull the brain out by cutting the optic and trigeminal nerves.</li>
	<li>Carefully transfer the brain onto a flat, clean glass surface for dissection on ice.</li>
	<li>Use a flat metal spatula and a razor blade to dissect the regions of interest of the brain. Place the tissues in a 2 mL centrifuge tube and weigh the tissue obtained. Use the brain section directly for cell isolation or quickly freeze it in liquid nitrogen for later use.</li>
</ol>
9. Cell isolation
<ol>
	<li>Make sure to work in a clean and sterile environment. Working under a class-II biosafety cabinet (BSC) is advised. Ensure that the gloves and every piece of equipment introduced into the BSC are sterile.</li>
	<li>To prepare the cells for cell sorting, follow the protocol from Jaclyn M. Schwarz14. 
	NOTE: In this experiment, a commercially available Neural dissociation kit (see Table of Materials) was used for cell preparation.
	<ol>
		<li>Put the samples into a 2 mL centrifuge tube with 1 mL of HBSS (Ca- and Mg-free), and then centrifuge (300 x g, 2 min, room temperature = RT) and remove the supernatant without disturbing the pellet.</li>
		<li>Add 1900 &#181;L of enzyme mix-1 and incubate it for 15 min at 37 &#176;C while agitating the tubes by inversion every 5 min.</li>
		<li>Add 30 &#181;L of enzyme mix-2, agitate gently with pipette 1 (see Table of Materials) back and forth 30 times. Then, incubate for 15 min at 37 &#176;C while agitating the tubes by inversion every 5 min.</li>
		<li>Mix gently back and forth with pipette 2, and then pipette 3 before incubating for 10 min at 37 &#176;C to dissociate the tissues.</li>
		<li>Filter the cells with an 80 &#181;m cell strainer, add 10 mL of HBSS (Ca- and Mg-free). Centrifuge (300 x g, 10 min, RT) and remove the supernatant without disturbing the pellet.</li>
		<li>For myelin depletion, resuspend the pellet with 400 &#181;L of myelin removal buffer (see Table of Materials), and then add 100 &#181;L of myelin removal beads (see Table of Materials), before incubating for 15 min at 4 &#176;C.</li>
		<li>Add 5 mL of myelin removal buffer, centrifuge (300 x g, 10 min, RT), and remove the supernatant without disturbing the pellet.</li>
		<li>Add 500 &#181;L of myelin removal buffer and place the tube into the magnetic field column. Wash the column with 1 mL of myelin removal buffer four times.</li>
		<li>Centrifuge (300 x g, 2 min, RT) and remove the supernatant without disturbing the pellet. Vortex briefly (2 s) to dissociate the cells and add 5 &#181;L of Fc block CD32. Vortex again and incubate for 5 min at 4 &#176;C.</li>
		<li>Add 100 &#181;L of the mix of primary antibodies of interest; incubate for 20 min at 4 &#176;C.</li>
		<li>Centrifuge (350 x g, 5 min, 4 &#176;C) and remove the supernatant without disturbing the pellet. Blot the tubes upside-down on soft paper.</li>
		<li>After a brief vortex (2 s), add 100 &#181;L of the secondary antibody mix and incubate for 15 min at 4 &#176;C.</li>
		<li>Add 2 mL of myelin removal buffer, centrifuge (350 x g, 5 min, 4 &#176;C) and remove the supernatant without disturbing the pellet. Blot the tubes upside-down on soft paper. Resuspend the cells in 250 &#181;L of sterile PBS and proceed directly to cell sorting.</li>
	</ol>
	</li>
</ol>
10. Cell sorting
<ol>
	<li>Prepare the microfuge tube with 500 &#181;L of sterile PBS for collecting the sorted cells.</li>
	<li>Add 10 &#181;L of Hoechst per 1000 &#181;L of cell solution to color the nuclei of the living cells and distinguish them from the dead cells.</li>
	<li>Transfer the cells as soon as possible to the cell sorting machine at 4 &#176;C.</li>
	<li>First, sort the cells by forward and side scatter, and then sort the Hoechst positive cells. Next, sort the cells based on the antibodies of interest. Collect the positively stained cells separately. Make sure to distinguish the positive cells from the autofluorescent ones.</li>
	<li>Count the number of sorted cells for each pool of interest.</li>
</ol>
11. Gamma counting
<ol>
	<li>Calibrate the &#947; counting system with 10 &#181;L of the same phantom solution used for the SPECT calibration but dilute it in 1 mL of water. 
	NOTE: The phantom solution is diluted because of the enhanced precision of the &#947; counting system.</li>
	<li>Place the tube of sorted cells in the &#947; counting system and proceed with the &#947; counting according to the manufacturer&#39;s protocol.</li>
</ol>

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The authors declare no conflicts of interest.

To our knowledge, this technique was the first to describe an approach that allows a better understanding of in vivo binding alterations of a radiotracer at the cellular level. The protocol describes a multiscale method to quantify radiotracer binding at the cellular level using [125I]CLINDE (TSPO) or [125I]R91150 (5HT2AR) as examples.
This technique is robust and sensitive enough to precisely detect the cellular origin of a wide spectrum of glial cell...

Neurodegenerative diseases, such as Alzheimer&#39;s Disease (AD), are characterized by a neuronal loss associated with increased symptoms. AD, the most common cause of dementia, accounting 60%-70% of cases, affects around 50 million people worldwide1. At a neuropathological level, the two major characteristics of AD are the accumulation of extracellular amyloid-&#946; (A&#946;) plaques and intracellular Tau neurofibrillary tangles. Glial cell alterations have also been associated with AD2 and possible disruption of several neurotransmitter systems3,4.
The TgF344-AD rat line has been modified to model AD by expressing human APP and PS1&#916;E9 transgenes, leading to soluble and insoluble A&#946;-40 and A&#946;-42 expression and amyloid plaque formation5. It also presents the accumulation of hyperphosphorylated forms of the Tau protein leading to tauopathy. From the age of 9-24 months, the rats progressively develop the pathological hallmarks of AD and a cognitive impairment5,6,7,8,9.
Positron Emission Tomography (PET), Single-Photon Emission computed Tomography (SPECT), and autoradiography are techniques based on the emission and quantification of &#947; rays. Radiotracers are quantified either in vivo (PET and SPECT) or ex vivo/in vitro (autoradiography). Those sensitive techniques have contributed to the understanding of mechanisms of several brain diseases, such as AD. Indeed, in terms of neuroinflammation, there are a lot of studies assessing 18 kDa Translocator Protein (TSPO), an in vivo neuroinflammation marker, with radiolabeled tracers such as [11C]-(R)-PK11195 or [11C]PBR28 (for review see10). In addition, alterations of neurotransmitter systems have been studied using radiotracers11,12,13.
However, those techniques do not determine the cellular origin of the radioactive signal. This could hamper the interpretation of the biological underpinnings of the alteration in the binding of a radioligand in PET/SPECT. For instance, in the case of TSPO studies of neuroinflammation, understanding whether the increase or decrease of TSPO is due to astrocytic or microglial changes is of paramount importance. The Fluorescence-Activated Cell Sorting to Radioligand Treated Tissue (FACS-RTT) technique was developed to get around these problems, allowing the assessment of radioligand binding in every cell type separately and the quantification of the target-protein density per cell. This innovative technique is consequently complementary and highly compatible with PET and SPECT imaging.
Here, this technique was applied along two axes: the study of neuroinflammation using TSPO-specific radioligands and assessing the serotonergic system. On the first axis, the aim was to understand the cellular origin of the TSPO signal in response to an acute inflammatory reaction. Therefore, FACS-RTT was used on the brain tissues of rats after the induction of neuroinflammation via a lipopolysaccharide (LPS) injection and following an in vivo [125I]CLINDE SPECT imaging study. Further, the same imaging and FACS-RTT protocol were applied on 12- and 24-month-old TgF344-AD rats and matching wild-type (WT) rats. The second axis aimed to determine the origin of serotoninergic system alterations in this rat model via ex vivo 5-HT2AR density assessment by cell type.

<table><tbody><tr><td>Acetic acid</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Acetonitrile</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>BioVet</td><td>BioVet</td><td></td><td>Software for vitals check</td></tr><tr><td>Bondclone C18 reverse-phase column</td><td>Phenomenex, Schlieren, Switzerland</td><td></td><td></td></tr><tr><td>Des-Sur</td><td>University Hospital of Geneva</td><td></td><td>Virucide</td></tr><tr><td>Fc Block / anti-CD32</td><td>BD Biosciences</td><td>BDB550270</td><td>Reactivity for rat</td></tr><tr><td>FITC-conjugated anti-rat CD90</td><td>Biolegend</td><td>202504</td><td>Reactivity for rat</td></tr><tr><td>Heparin</td><td>B. Braun</td><td>B01AB01</td><td></td></tr><tr><td>HPLC</td><td>Knauer</td><td></td><td></td></tr><tr><td>Insyte-W 24 GA 0.75 IN 0.7 x 19 mm</td><td>BD Biosciences</td><td>321312</td><td>24 G catheter</td></tr><tr><td>Isoflurane</td><td>Baxter</td><td>ZDG9623</td><td></td></tr><tr><td>Lacryvisc</td><td>Alcon</td><td>2160699</td><td></td></tr><tr><td>LS Columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>MACS MultiStand</td><td>Miltenyi Biotec</td><td>130-042-303</td><td></td></tr><tr><td>Micropore soft tape</td><td>3M</td><td>F51DA01</td><td></td></tr><tr><td>MILabs-Uspect II</td><td>MILabs</td><td></td><td>Software for SPECT Camera</td></tr><tr><td>MoFlo Astrios</td><td>Beckman Coulter</td><td></td><td>Cell sorter</td></tr><tr><td>Myelin Removal Beads II</td><td>Miltenyi Biotec</td><td>130-096-733</td><td>Contains beads and myelin removal buffer.</td></tr><tr><td>NaCl 0.9% Sterile solution</td><td>B. Braun</td><td>395202</td><td></td></tr><tr><td>Neural Dissociation Kit (P)</td><td>Miltenyi Biotec</td><td>130-092-628</td><td>Contains the enzyme mixes, pipets 1, 2 and 3.</td></tr><tr><td>Nylon Mesh Sheet</td><td>Amazon</td><td>CMN-0074-10YD</td><td>40 inch width, 80 micron size mesh</td></tr><tr><td>Peracetic acid</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>QuadroMACS Separator</td><td>Miltenyi Biotec</td><td>130-090-976</td><td></td></tr><tr><td>R91150 pr&amp;eacute;cursor</td><td>CERMN</td><td></td><td></td></tr><tr><td>Sep-Pak C18 Column</td><td>Waters</td><td></td><td>Concentration column</td></tr><tr><td>Sodium iodide Na&lt;sup&gt;125&lt;/sup&gt;</td><td>PerkinElmer</td><td></td><td></td></tr><tr><td>Tributylin precursor</td><td>CERMN</td><td></td><td></td></tr><tr><td>U-SPECT Rec2.38c</td><td>MILabs</td><td>Version Rec2.38c</td><td>Software for SPECT images reconstruction</td></tr><tr><td>USPECT II</td><td>MILabs</td><td></td><td>Spect Camera</td></tr><tr><td>Wizard 3&quot;</td><td>PerkinElmer</td><td></td><td>Gamma counter</td></tr></tbody></table>

fluorescence-activated cell sorting-radioligand treated tissue (facs-rtt) to determine the cellular origin of radioactive signal

Glial cells probably have a considerable implication in the pathophysiology of neurodegenerative disorders, such as Alzheimer's disease (AD). Their alterations are perhaps associated with a pro-inflammatory state. The TgF344-AD rat strain has been designed to express human APP and human PS1&#916;E9 genes, encoding for amyloid proteins A&#946;-40 and A&#946;-42 and displays amyloid pathology and cognitive deficits with aging. The TgF344-AD rat model is used in this study to evaluate the cellular origin of the 18 kDa translocator protein (TSPO, a marker of glial cell activation) binding, and the 5HT2A-receptor (5HT2AR) serotonin receptor levels that are possibly disrupted in AD. The technique presented here is Fluorescence-Activated Cell Sorting to Radioligand Treated Tissue (FACS-RTT), a quantitative cell-type-specific technique complementary to in vivo PET or SPECT or ex vivo/in vitro autoradiography techniques. It quantifies the same radiolabeled tracer used prior for imaging, using a &#947; counter after cytometry cell sorting. This allows determining the cellular origin of the radiolabeled protein with high cellular specificity and sensitivity. For example, studies with FACS-RTT showed that (i) the increase in TSPO binding was associated with microglia in a rat model of lipopolysaccharide (LPS)-induced neuroinflammation, (ii) an increase in TSPO binding at 12- and 18-months was associated with astrocytes first, and then microglia in the TgF344-AD rats compared to wild type (WT) rats, and (iii) the striatal density of 5HT2AR decreases in astrocytes at 18 months in the same rat AD model. Interestingly, this technique can be extended to virtually all radiotracers.

WT rats experienced in vivo SPECT scan with [125I]CLINDE radiotracer after a unilateral LPS injection (Figure 2). This scan (using summed data from images of 45-60 min post radiotracer injection) showed higher binding of [125I]CLINDE in the site of the LPS injection (Figure 2A) than in the contralateral region of the brain (Figure 2B). The ex vivo samples that underwent FACS-RTT confirmed thos...

Watch this Scientific Journal Video about Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue FACS-RTT to Determine the Cellular Origin of Radioactive Signal at JoVE.com

Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue FACS-RTT to Determine the Cellular Origin of Radioactive Signal

Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue (FACS-RTT) is a powerful tool to study the role of the 18 kDa translocator protein or Serotonin 5HT2A-receptor expression in Alzheimer&#39;s Disease at a cellular scale. This protocol describes the ex-vivo application of FACS-RTT in the TgF344-AD rat model.

Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue (FACS-RTT) to Determine the Cellular Origin of Radioactive Signal

Glial cells probably have a considerable implication in the pathophysiology of neurodegenerative disorders, such as Alzheimer's disease (AD). Their ...

fluorescence-activated-cell-sorting-radioligand-treated-tissue-facs

Research

JoVE Journal

Neuroscience

2.3K Views.  University Hospitals of Geneva. Dit protocol is complementair aan in vivo beeldvorming. Het maakt celtype en cellulaire niveau kwantificering van radiotracers mogelijk, wat een andere schaal is dan degene die in vivo is verkregen. Het belangrijkste voordeel van deze techniek is de gevoeligheid en de cellulaire resolutie die wordt gegeven door het gebruik van radioliganden en de celsortering.Schakel de camera in en laad de bedieningssoftware. Klik op de Home XYZ stage knop om homing uit te voeren. Stel het experiment ...

Video: Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue FACS-RTT to Determine the Cellular Origin of Radioactive Signal

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Cancer Research

Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light absorption by tissue chromophores (e.g., hemoglobin, water, lipids, melanin) typically limits optical signal propagation through thicknesses larger than a few millimeters2. Compared to other visible wavelengths, tissue absorption for red and near-infrared (near-IR) light absorption dramatically decreases and non-elastic scattering becomes the dominant light-tissue interaction mechanism. The relatively recent development of fluorescent agents that absorb and emit light in the near-IR range (600-1000 nm), has driven the development of imaging systems and light propagation models that can achieve whole body three-dimensional imaging in small animals3.
Despite great strides in this area, the ill-posed nature of diffuse fluorescence tomography remains a significant problem for the stability, contrast recovery and spatial resolution of image reconstruction techniques and the optimal approach to FMI in small animals has yet to be agreed on. The majority of research groups have invested in charge-coupled device (CCD)-based systems that provide abundant tissue-sampling but suboptimal sensitivity4-9, while our group and a few others10-13 have pursued systems based on very high sensitivity detectors, that at this time allow dense tissue sampling to be achieved only at the cost of low imaging throughput. Here we demonstrate the methodology for applying single-photon detection technology in a fluorescence tomography system to localize a cancerous brain lesion in a mouse model.
The fluorescence tomography (FT) system employed single photon counting using photomultiplier tubes (PMT) and information-rich time-domain light detection in a non-contact conformation11. This provides a simultaneous collection of transmitted excitation and emission light, and includes automatic fluorescence excitation exposure control14, laser referencing, and co-registration with a small animal computed tomography (microCT) system15. A nude mouse model was used for imaging. The animal was inoculated orthotopically with a human glioma cell line (U251) in the left cerebral hemisphere and imaged 2 weeks later. The tumor was made to fluoresce by injecting a fluorescent tracer, IRDye 800CW-EGF (LI-COR Biosciences, Lincoln, NE) targeted to epidermal growth factor receptor, a cell membrane protein known to be overexpressed in the U251 tumor line and many other cancers18. A second, untargeted fluorescent tracer, Alexa Fluor 647 (Life Technologies, Grand Island, NY) was also injected to account for non-receptor mediated effects on the uptake of the targeted tracers to provide a means of quantifying tracer binding and receptor availability/density27. A CT-guided, time-domain algorithm was used to reconstruct the location of both fluorescent tracers (i.e., the location of the tumor) in the mouse brain and their ability to localize the tumor was verified by contrast-enhanced magnetic resonance imaging.
Though demonstrated for fluorescence imaging in a glioma mouse model, the methodology presented in this video can be extended to different tumor models in various small animal models potentially up to the size of a rat17.

Diffuse fluorescence tomography offers a relatively low-cost and potentially high-throughout approach to preclinical in vivo tumor imaging. The methodology of optical data collection, calibration, and image reconstruction is presented for a computed tomography-guided non-contact time-domain system using fluorescent targeting of the tumor biomarker epidermal growth factor receptor in a mouse glioma model.

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light ...

Medicine

Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that participate in regenerating tissue, tumor formation, or vasculogenesis, we devised a multi-colored cellular transplant model of tumor development in which cell populations originate from different fluorescently colored reporter gene mice and are transplanted, engrafted or injected in and around a developing tumor. These colored cells are then recruited and incorporated into the tumor stroma. In order to quantitatively assess bone marrow derived tumor stromal cells, we transplanted GFP expressing transgenic whole bone marrow into lethally irradiated RFP expressing mice as approved by IACUC. 0ovarian tumors that were orthotopically injected into the transplanted mice were excised 6-8 weeks post engraftment and analyzed for bone marrow marker of origin (GFP) as well as antibody markers to detect tumor associated stroma using multispectral imaging techniques. We then adapted a methodology we call MIMicc- Multispectral Interrogation of Multiplexed cellular compositions, using multispectral unmixing of fluoroprobes to quantitatively assess which labeled cell came from which starting populations (based on original reporter gene labels), and as our ability to unmix 4, 5, 6 or more spectra per slide increases, we&#39;ve added additional immunohistochemistry associated with cell lineages or differentiation to increase precision. Utilizing software to detect co-localized multiplexed-fluorescent signals, tumor stromal populations can be traced, enumerated and characterized based on marker staining.1

Complex tissue masses, from organs to tumors, are composed of various cellular elements. We elucidated the contribution of cellular phenotypes within a tissue utilizing multi-labeled fluorescent transgenic mice in combination with multiparameter immunofluorescent staining followed by spectral unmixing to decipher cell origin as well as cell characteristics based on protein expression.

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that ...

Positron Emission Tomography Imaging of Cell Trafficking: A Method of Cell Radiolabeling

Stem cell and chimeric antigen receptor (CAR) T-cell therapies are emerging as promising therapeutics for organ regeneration and as immunotherapy for various cancers. Despite significant progress having been made in these areas, there is still more to be learned to better understand the pharmacokinetics and pharmacodynamics of the administered therapeutic cells in the living system. For noninvasive, in vivo tracking of cells with positron emission tomography (PET), a novel [89Zr]Zr-p-isothiocyanatobenzyl-desferrioxamine ([89Zr]Zr-DBN)-mediated cell radiolabeling method has been developed utilizing 89Zr (t1/2 78.4 h). The present protocol describes a [89Zr]Zr-DBN-mediated, ready-to-use, radiolabeling synthon for direct radiolabeling of variety of cells, including mesenchymal stem cells, lineage-guided cardiopoietic stem cells, liver regenerating hepatocytes, white blood cells, melanoma cells, and dendritic cells. The developed methodology enables noninvasive PET imaging of cell trafficking for up to 7 days post-administration without affecting the nature or the function of the radiolabeled cells. Additionally, this protocol describes a stepwise method for the radiosynthesis of [89Zr]Zr-DBN, biocompatible formulation of [89Zr]Zr-DBN, preparation of cells for radiolabeling, and finally the radiolabeling of cells with [89Zr]Zr-DBN, including all the intricate details needed for the successful radiolabeling of cells.

Presented here is a protocol to radiolabel cells with a positron emission tomography (PET) radioisotope, 89Zr (t1/2 78.4 h), using a ready-to-use radiolabeling synthon, [89Zr]Zr-p-isothiocyanatobenzyl-desferrioxamine ([89Zr]Zr-DBN). Radiolabeling cells with [89Zr]Zr-DBN allows noninvasive tracking and imaging of administered radiolabeled cells in the body with PET for up to 7 days post-administration.

Stem cell and chimeric antigen receptor (CAR) T-cell therapies are emerging as promising therapeutics for organ regeneration and as immunotherapy for ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Immunology and Infection

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific sets of sparsely distributed activated neurons called neuronal ensembles that mediate learned associations. Fluorescence Activated Cell Sorting (FACS) has recently been optimized for adult rat brain tissue and allowed isolation of activated neurons using antibodies against the neuronal marker NeuN and Fos protein, a marker of strongly activated neurons. Until now, Fos-expressing neurons and other cell types were isolated from fresh tissue, which entailed long processing days and allowed very limited numbers of brain samples to be assessed after lengthy and complex behavioral procedures. Here we found that yields of Fos-expressing neurons and Fos mRNA from dorsal striatum were similar between freshly dissected tissue and tissue frozen at -80 &#186;C for 3 - 21 days. In addition, we confirmed the phenotype of the NeuN-positive and NeuN-negative sorted cells by assessing gene expression of neuronal (NeuN), astrocytic (GFAP), oligodendrocytic (Oligo2) and microgial (Iba1) markers, which indicates that frozen tissue can also be used for FACS isolation of glial cell types. Overall, it is possible to collect, dissect and freeze brain tissue for multiple FACS sessions. This maximizes the amount of data obtained from valuable animal subjects that have often undergone long and complex behavioral procedures.

Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

Here we present a Fluorescence Activated Cell Sorting (FACS) protocol to study molecular alterations in Fos-expressing neuronal ensembles from both fresh and frozen brain tissue. The use of frozen tissue allows FACS isolation of many brain areas over multiple sessions to maximize the use of valuable animal subjects.

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific ...

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

Biology

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample that can provide information about antigen coexpression and spatial distribution of the markers. However, a lack of suitable antibodies for formalin-fixed tissues may restrict the nature of markers that can be detected. In addition, the staining method is time-consuming. Here we describe a rapid method to perform multispectral fluorescence imaging on frozen tissues. The method includes the fluorophore combinations used, detailed steps for the staining of mouse and human frozen tissues, and the scanning, acquisition, and analysis procedures. For staining analysis, a commercially available semiautomated multispectral fluorescence imaging system is used. Through this method, up to six different markers were stained and detected in a single frozen tissue section. The machine learning analysis software can phenotype cells that can be used for quantitative analysis. The method described here for frozen tissues is useful for the detection of markers that cannot be detected in FFPE tissues or for which antibodies are not available for FFPE tissues.

We describe a rapid staining method to perform multispectral imaging on frozen tissues.

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample ...

Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue (FACS-RTT) to Determine the Cellular Origin of Radioactive Signal

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