Name: radionuclide-fluorescence reporter gene imaging to track tumor progression in rodent tumor models
Uploaded: 2018-03-13T14:00:00
Duration: 10 min 4 s
Description: Watch this Scientific Journal Video about Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models at JoVE.com

The research was supported by the King&#39;s College London and UCL Comprehensive Cancer Imaging Centre, funded by Cancer Research UK and EPSRC in association with the MRC and DoH (England); the National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy&#39;s and St Thomas&#39; NHS Foundation Trust and King&#39;s College London; the Centre of Excellence in Medical Engineering funded by the Wellcome Trust and EPSRC under grant number WT 088641/Z/09/Z; a Cancer Research UK Multidisciplinary Project Award to GOF and PJB, and a King&#39;s Health Partners grant to GOF. The nanoPET/CT and nanoSPECT/CT scanners were purchased and maintained by an equipment grant from the Wellcome Trust. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, or the DoH.

The authors declare that they have no competing financial interests.

The first step to render cancer cells traceable in vivo by this method requires engineering them to express the NIS-FP fusion reporter. The choice of the fluorescent protein in the fusion reporter is critical as oligomerizing fluorescent proteins can lead to artificial reporter clustering, thereby negatively affecting its function. We have had success with proven monomeric fluorescent proteins such as mEGFP (with the monomerizing mutation A206K36,37), mTagRFP, or mCherry. NIS can either be of human or mouse origin (hNIS or msNIS) depending on the purpose of the experiment and the cancer model. Transduction efficiencies generally vary between different cancer cell lines. However, generated cancer cell lines are subsequently purified by FACS in this protocol, thereby reducing the need for optimizing transduction conditions. Transduction with high multiplicity of infection is not always advisable as multiple construct integration into the genome is likely to result not only in higher construct expression but also in more unwanted/unregulated genome modification. Therefore, it is important to let polyclonal transduced cells grow to stability of expression (monitored by flow cytometry) and avoid sorting the brightest clones only by FACS. It also renders functional validation of non-reporter features crucial before these cells should be used for in vivo experiments. A recently developed alternative to viral gene delivery is gene editing technology38, which offers more specific control over viral integration sites. Expression analysis by flow cytometry and immunoblotting is important. Flow cytometry allows acquisition of population-based single cell data, for example to examine whether there is any drift in reporter expression levels over time. It relies on the FP moiety only, unless cells are also stained with an antibody directed against surface or total NIS. Flow cytometry does not inform on fusion reporter integrity. In contrast, immunoblotting reports on the integrity of the fusion reporter. The molecular weight of NIS and the FP must be added to determine the expected molecular weight of the chosen NIS-FP. Confocal fluorescence microscopy demonstrated fusion reporter colocalization with the plasma-membrane marker wheat germ agglutinin in all newly made cell lines. This was the expected cellular location for most of the protein and indicated a go-ahead milestone for subsequent functional validation. If minimal/no NIS-FP was found on the plasma membrane (e.g. only in internal cellular compartments), this would indicate a cell biological issue with the fusion reporter in this cell line, or a potential mutation of the fusion reporter affecting its intracellular trafficking. It is noteworthy that we have not observed such a case in any of the cancer cells we tested so far, which included: A375P, A375M2, SK-Mel28, WM983A/B (human melanoma); MCF-7, MDA-MB-231, MDA-MB-436 (human breast cancer); NCI-H1975 (human lung cancer); SK-Hep1 (human liver cancer); 4T1, 4T1.2, 66cl4, 67NR, FARN168 (murine inflammatory breast cancer); B16F0, B16F3, B16F10 (murine melanoma); MTLn3 (rat breast adenocarcinoma).
NIS function must be measured using uptake assays with radioactive NIS substrates. Due to the SPECT radiotracer 99mTcO4- being generator-produced and therefore widely available in hospitals without the need for any radiotracer synthesis as well as having a more convenient longer half-life (6.01 h for 99mTc as compared to 110 min for 18F), we used this NIS substrate for routine functional validation of new NIS-FP expressing cell lines. Pre-blocking of NIS-expressing cells with the NIS co-substrate sodium perchlorate resulted in the expected reduction/abolishment of radiotracer uptake, thereby demonstrating specificity of radiotracer uptake. This NIS specificity test is a critical validation step. If a NIS specificity experiment would not result in reduced radiotracer uptake comparable to the respective parental cells, either a technical error during the experiment has occurred, or the radiotracer uptake was not due to NIS. It is also possible that sodium perchlorate pre-blocking reduces radiotracer uptake in a parental cell line; this would identify cell lines with endogenous functional NIS expression (e.g. stimulated thyroid cells6).
A crucial advantage of this imaging protocol is that information is collected in 3D and over time. This allows the comparison of images from the same animal over time, thereby providing paired data and thus overcoming the issues caused by inter-animal variability. This contrasts with most non-imaging related metastasis assessment methods that are based on sacrificing different animals at different time points. In Figure 3B it is evident how metastatic spread and outgrowth progressed over time in an individual animal. The signals detected by PET/CT imaging are fundamentally caused by NIS expression. This includes all signals from exogenously NIS-expressing cancer cells as well as all organs endogenously expressing NIS. Typical endogenous NIS signals are found in thyroid and salivary glands, the stomach, and, at low levels in some parts of the mammary and lachrymal glands. In addition to endogenous NIS expression, the NIS radiotracer [18F]BF4- is also excreted via the kidneys, thereby explaining radiotracer uptake in urine-filled bladders. Kidney uptake is no longer detectable at the imaging time point recommended in this protocol (45 min post radiotracer injection6). If signals from urine-filled bladders should lead to signal-to-background issues, the bladder can be mechanically emptied under anesthesia before imaging. Importantly, the endogenous signals can vary between animal strains. It is also noteworthy that endogenous NIS expression in the mammary glands can be higher under lactating conditions10. In the presented case and in the cases of those metastatic cell lines successfully characterized before (cf. list above), we did not find endogenous NIS expression to significantly interfere with metastasis detection. It is noteworthy, that [18F]BF4- remains more available for uptake into cancerous tissues as compared to iodide, because iodide is metabolized into thyroid hormones6. This phenomenon might also contribute to larger amounts of radioiodide in the blood stream as compared to [18F]BF4- 6. For different applications (cancer cell tracking in other cancers or non-cancer cell tracking applications), this might differ, and it is therefore recommended to assess whether endogenous NIS expression is likely to cause signal-to-background issues through preliminary experiments. An important aspect in preclinical imaging is the molar activity of the radiotracer. The method described here uses ~1.5 GBq 18F- as starting material14 and has been shown to produce molar activities significantly above the previously reported substitution method12. [18F]BF4- produced at molar activities &#8804;1 GBq/&#181;mol12 can lead to reduced uptake in NIS-expressing tissues. This is of particular importance when the injected amount of radioactivity per kilogram is high, i.e. when small animals such as mice are imaged39; it is less important in the human setting40. High molar activities are therefore imperative for high-quality preclinical PET imaging. Molar activities obtained by the boron trifluoride addition method14, which is shown in its automated form in this protocol, overcome this issue. Furthermore, it is noteworthy that the presented protocol for [18F]BF4- synthesis is not compliant with good manufacturing practice (GMP) and therefore unsuitable for use in human clinical trials in this form. A GMP protocol (via the 18F substitution method to radiolabel BF4-) is available elsewhere40.
PET/CT imaging allows the visualization of radiotracer uptake, which is indicative of NIS-mediated radiotracer uptake stemming from NIS-FP expressing cancer cells. More importantly, the associated PET signals can be quantified. It is necessary to apply reliable thresholding procedures to ensure a consistent and unbiased differentiation of relevant signals from any potential background. As the background varies in different locations in vivo, it is important to consider local/regional thresholding and segmentation. One such method was developed by and named after Otsu34, and its 3D implementation is employed for 3D rendering of the primary tumor and metastases in this protocol. Generally, the image seen by the observer visually corresponds best to the quantified %injected dose (%ID) values. As for image-based quantification, it is also important to normalize the measured radioactivity values of the different tissues to their volumes. There are two predominantly used ways of expressing normalized results, (i) %ID per volume (e.g. %ID/mL), and (ii) standard uptake value (SUV35). They differ in that %ID/mL takes into account the individual volume only, while SUV is a measure that is relative to the average radioactivity across the whole animal. It is also important to note that NIS imaging renders the live tumor volume (LTV) accessible, because dead/dying cells not synthesizing ATP can no longer import radiotracer10. This explains the large low-signal area within the primary tumor (&#34;donut shaped&#34; tumor) indicating areas of tumor cell death/necrosis. Importantly, LTV was a much more reliable measure of tumor burden as compared to the crude tumor volume accessible by caliper measurements (which does not take into account viability and assesses only superficial tumor regions).
A major advantage of this dual-mode tracking strategy is evident when harvesting tissues after animal culling. Guided by in vivo images and assisted by fluorescent cancer cells during animal dissection, small and deep-seated organs/metastases can also be reliably harvested. Frozen tissue preservation/sectioning methodology enables the direct fluorescence imaging of GFP without the need for staining with an anti-GFP antibody, but at the expense of reduced structural tissue preservation as compared to formalin-fixed paraffin-embedded methodology (FFPE). The latter critically requires also anti-FP staining, because the FFPE method is incompatible with intact preservation of fluorescent proteins (due to fixation/dehydration/rehydration). While the fluorescence signal is indicative of tumor cell presence, it is important to ensure this classification is confirmed by ex vivo radioactivity measurements of the harvested tissues (&#39;biodistribution&#39;). Ex vivo radioactivity measurements are more sensitive than visual detection of fluorescence, hence can allow the identification of cancer cell-dependent signals that would otherwise remain undetected. In the case of a terminal imaging session, it is critical to accurately note the injected radiotracer amounts as well as the times of radiotracer radioactivity measurements, animal injection, animal culling, and calibrated scintillation counter measurements of harvested tissues. This is crucial to ensure correction for radiotracer decay and thereby enable reliable biodistribution analysis.
PET/CT imaging enables repeated non-invasive 3D quantification of tumor progression including the assessment of metastatic spread on a whole-body level. This feature is a significant advantage over conventional methods, which often rely on large cohorts of animals that are euthanized for the assessment of tumor progression at varying time points. The advantages of this imaging-based approach are: (i) highly sensitive non-invasive 3D in vivo quantification, (ii) a significant reduction in animal numbers due to the possibility of repeat imaging, (iii) the acquisition of longitudinal paired data from subsequent imaging sessions improving statistics by excluding inter-animal variability, which in turn further reduces animal numbers, (iv) automated production of [18F]BF4- at high specific activities, and (v) the intrinsic option for ex vivo confirmation in tissues by fluorescence methodologies such as microscopy or cytometry.
In vivo cell tracking is a growing field. It has been fueled by recent advancements in imaging technology, which resulted in enhanced resolution, detection limits and multiplex capability (via multi-modal imaging). In this protocol, we apply this concept to track tumor progression including spontaneous cancer cell metastasis in 3D by repeat imaging. Applications include studies aimed at unraveling the mechanisms of spontaneous cancer cell metastasis. For example, traceable tumor cells could be used to study the impact of different immune cell components (as present/functional in animal strains of different levels of immunocompromisation) on the metastatic process. Similarly, the impact of individual genes, either in the animal strain or the cancer cell line, could be studied. Furthermore, the presented protocol could be used to assess/validate the efficacy of specific drugs or therapeutic concepts on tumor progression. Importantly, this reporter gene:radiotracer pair for PET imaging (NIS:[18F]BF4-) could also be used for different cell tracking applications. For example, several cell therapies are currently emerging as promising therapeutic approaches. This includes cellular therapeutics for cancer treatment41 but also in transplantation42 and regenerative medicine43,44 settings. Whole-body in vivo cell tracking is becoming increasingly important for the development and clinical translation of cellular therapeutics, for example, for evaluating safety and for therapy monitoring.

Metastatic disease is the cause for most cancer-related deaths 1. Despite extensive research into metastatic processes, reliable monitoring of cancer metastasis in animal model systems is difficult to achieve. Recent advances in whole-body imaging technologies and multi-modal imaging approaches have enabled non-invasive in vivo cell tracking2,3,4,5. The latter can be used as a tool to monitor the presence, distribution, quantity, and viability of cells, non-invasively and repeatedly in a live animal or a human.
The purpose of the method described here is to longitudinally and non-invasively track cancer cells in 3D in living rodent tumor models. Using this method, researchers will be able to accurately quantify tumor progression including metastatic spread in 3D. Compared to traditional non-imaging-based techniques, this method offers the acquisition of quantitative data with largely reduced animal numbers. Another feature of this method is that it allows correlation of in vivo imaging with streamlined downstream ex vivo analysis of the tracked cells in harvested tissues by histology or cytometry3,6.
The rationale for the development of this method was to provide an in vivo tool for the monitoring and quantification of the whole metastatic process in rodent tumor models. Importantly, it was designed to minimize animal use while at the same time reducing inter-animal variability. Longitudinal non-invasive whole-body imaging is excellently suited to inform on metastatic outgrowth, for which per se it is difficult to predict accurately the time and location of its occurrence. Whole-body 3D imaging has therefore been at the center of method development. To close the scale gap between whole-body in vivo imaging and potential downstream ex vivo histological confirmation, a multi-scale imaging approach based on a dual-mode radionuclide-fluorescence reporter was adopted3,6.
Positron emission tomography (PET) is the most sensitive 3D whole-body imaging technology currently available offering excellent depth penetration and absolute quantification7 with a resolution of &#60;1 mm8,9. Currently, preclinical cell tracking on the whole-body level by radionuclide imaging can reliably detect cells at densities of ~1,000 cells per volume of a million cells3,6 with resolutions in the sub-millimeter region. Unlike intravital microscopy, it cannot detect single spreading cancer cells, but it does not require surgical procedures (e.g. window chambers), is not limited to a small field of view, and not by low tissue penetration and scatter. Bioluminescence imaging provides an inexpensive alternative, but is associated with scatter and light absorption issues as well as poor depth penetration, and consequently severely limited in quantification2. Fluorescence whole-body imaging has been used for the acquisition of 3D images, but it is much less sensitive compared to bioluminescence or radionuclide technologies2. Nonetheless, fluorescence offers the opportunity to perform ex vivo downstream tissue analysis by cytometry or microscopy. The latter closes the scale-gap between macroscopic whole-body imaging (mm resolution) and fluorescence microscopic tissue analysis (&#181;m resolution)3. Therefore, radionuclide and fluorescence modalities complement each other, ranging from the whole-body level to the (sub-)cellular scale.
Reporter gene imaging is ideally suited for prolonged cell tracking as required in metastasis research. In this application it is superior to direct cell labeling as it is (i) not affected by label dilution and thus not limited in tracking time, and (ii) better reflects live cell numbers. Consequently, whole-body cell tracking is particularly useful for applications in which traceable cells proliferate or expand in vivo, for example in cancer research3,6, for the detection of teratoma formation in stem cell research, or for the quantification of immune cell expansion5.
Various radionuclide-based reporter genes are available2. These include enzymes such as the herpes simplex virus HSV11 thymidine kinase (HSV1-tk), transporters such as the sodium iodide symporter (NIS) or the norepinephrine transporter (NET), as well as cell surface receptors such as the dopamine D2 receptor (D2R). NIS is a glycosylated trans-membrane protein that actively mediates iodide uptake, for example in follicular cells in the thyroid gland for the subsequent synthesis of thyroid hormones10. This process is driven by the symport of Na+ and relies on the cellular sodium gradient, which is maintained by the Na+/K+-ATPase11. Consequently, NIS better reflects living cell numbers than other reporters as iodide/radiotracer uptake is linked to an active Na+/K+ gradient rather than the mere presence of the transporter. Traditionally, radioiodide has been used for NIS imaging. For cell tracking, alternative NIS radiotracers that are not metabolically entrapped in the thyroid have been reported to be superior6. This recently developed PET radiotracer [18F]tetrafluoroborate ([18F]BF4-)12,13 shows superior pharmacokinetics as compared to radioiodide6 while being available at high specific activities14 without the need for complex radiochemistry facilities. [18F]BF4- can be synthesized via two different ways. The first method is based on isotope exchange of non-radioactive 19F in BF4- with radioactive 18F12. The second method is through addition of 18F to non-radioactive boron trifluoride14. The latter method was reported to yield higher specific activities14 and is the method of choice for preclinical imaging.
NIS is highly expressed in the thyroid tissues. It is also expressed in the salivary, lachrymal and lactating mammary glands as well as the stomach, but at lower levels as compared to the thyroid gland10. Therefore, excellent contrast imaging in other body regions can be achieved using NIS. It is also highly homologous between human, rat and mouse10. Moreover, there are no reports of toxicity upon ectopic NIS expression in non-thyroidal cells. Importantly, NIS has also not been associated with host immune responses, neither in humans nor in rodents. NIS has been used as a reporter gene to measure promoter activity15,16,17 and gene expression18,19,20,21,22,23 in several different contexts. It has also been used for non-invasive imaging of gene therapy vectors24,25, and to track cells in cardiac4, hematopoietic26, inflammation5, and neural studies27. Recently, NIS has also been used as a reporter gene to track cancer metastasis in vivo3,6.
In summary, the main advantages of this method over previous techniques are: (i) highly sensitive non-invasive 3D in vivo localization and quantification of metastatic spread, (ii) automated production of [18F]BF4- at high molar activities, (iii) a significant reduction in required animals through longitudinal imaging, (iv) the acquisition of paired data from subsequent imaging sessions resulting in improved statistical data, which in turn further reduces animal use, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by cytometry or fluorescence microscopy.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Step 1) Engineering and characterization&#160; of cancer cells to express the radionuclide-fluorescnece fusion reporter NIS-FP.</td>
		</tr>
		<tr>
			<td>2&#39;-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5&#39;-Bi-1H-benzimidazole</td>
			<td>Thermo Scientific</td>
			<td>H3570</td>
			<td>Trivial name: Hoechst 33342; CAS number: 23491-52-3; Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in Water.</td>
		</tr>
		<tr>
			<td>4T1 murine breast cancer cell line</td>
			<td>ATCC</td>
			<td>CRl-2539</td>
			<td>for details see ATCC website</td>
		</tr>
		<tr>
			<td>Automatic Cell Counter, e.g. CASYCounter</td>
			<td>Roche Diagnostics GmbH</td>
			<td>5651697001</td>
			<td>CASY Model TT Cell Counter and Analyzer</td>
		</tr>
		<tr>
			<td>CASYclean Cleaning Reagent</td>
			<td>Sedna Scientific</td>
			<td>2501036</td>
			<td></td>
		</tr>
		<tr>
			<td>CASYton Isotonic Diluent</td>
			<td>Sedna Scientific</td>
			<td>2501037</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Fluorescence Microscope, e.g. Leica TCS SP5</td>
			<td>Leica, Wetzlar, Germany</td>
			<td></td>
			<td>Equipped with Plan-Neofluor 25&#215;0.5NA and Plan-Apochromat 63&#215;1.4NA oil UV objectives and Diode (405 nm), Argon-ion (458, 477, 488, 496, 514 nm) and HeNe (543 and 633 nm) lasers; A Leica LAS AF Lite Software 4.0.11706 (Leica Microsystems CMS GmbH) was used for image acquisition and and anaysis</td>
		</tr>
		<tr>
			<td>Cover slips No. 1.5 thickness</td>
			<td>VWR International</td>
			<td>631-0150</td>
			<td></td>
		</tr>
		<tr>
			<td>Dabco</td>
			<td>Sigma</td>
			<td>290734</td>
			<td>Stock 125 mg/mL</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma</td>
			<td>D5546</td>
			<td>Supplement with 10 % (v/v) FBS and L-glutamine (2 mM) to make up the optimal growth medium for MDA-MB-231 cells.</td>
		</tr>
		<tr>
			<td>FACS sorter, e.g. BD FACSAria III&#160;</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Equipped with a BD FACS DIVA Software, a 6 Laser System (375/405/488/561/633 nm lasers) - cells sorted with a 100 &#956;m nozzle under 20 psi flow pressure, window extension of 2.0 &#956;m, 2.0 Neutral Density Filter and 3 kV plate voltage</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Sigma</td>
			<td>F9665</td>
			<td>Heat inactivated at 56 &#176;C for 30 min</td>
		</tr>
		<tr>
			<td>Automated Gamma Counter, e.g. 1282 Compugamma</td>
			<td>LKB Wallac Laboratory</td>
			<td></td>
			<td>99mTc-pertechnetate energy window 110-155 keV&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 18F energy window 175-220 keV</td>
		</tr>
		<tr>
			<td>Hoechst 33342 solution</td>
			<td>Life Technologies</td>
			<td>H1399</td>
			<td>1-3 &#181;g/mL; DAPI (from various supplieres) can be used instead.</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td>Solution 200 mM concentrated, sterile-filtered</td>
		</tr>
		<tr>
			<td>Linear polyethylenimine (PEI)</td>
			<td>Polyscience</td>
			<td>23966-2</td>
			<td>Linear, 25 kDa; transfection reagent for 293T cell line.</td>
		</tr>
		<tr>
			<td>MDA-MB-231 human breast cancer cells</td>
			<td>ATCC</td>
			<td>HTB-26</td>
			<td>for details see ATCC website</td>
		</tr>
		<tr>
			<td>Mowiol 4-88</td>
			<td>Sigma</td>
			<td>81381</td>
			<td></td>
		</tr>
		<tr>
			<td>pLNT SFFV NIS-mEGFP</td>
			<td>request from our lab</td>
			<td>n/a</td>
			<td>For details (generation and maps) see Supplementary Information</td>
		</tr>
		<tr>
			<td>pLNT SFFV NIS-mCherry</td>
			<td>request from our lab</td>
			<td>n/a</td>
			<td>For details (generation and maps) see Supplementary Information</td>
		</tr>
		<tr>
			<td>pMD2.G</td>
			<td>Addgene</td>
			<td>#12259</td>
			<td>plasmids encoding for the VSV-G envelope</td>
		</tr>
		<tr>
			<td>pRRE</td>
			<td>Addgene</td>
			<td>#12251</td>
			<td>packaging plasmid</td>
		</tr>
		<tr>
			<td>pRSV-Rev</td>
			<td>Addgene</td>
			<td>#12253</td>
			<td>packaging plasmid</td>
		</tr>
		<tr>
			<td>Paraformaldehyde solution 4 % (w/v) in PBS</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-281692</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma</td>
			<td>P43330</td>
			<td>Containing penicillin (10,000 units/mL) and streptomycin (10 mg/mL), sterile-filtered</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td>pH 7.4, sterile-filtered and without calcium chloride and magnesium chloride</td>
		</tr>
		<tr>
			<td>Poly(vinyl alcohol - vinyl acetate)</td>
			<td>Polysciences</td>
			<td>17951</td>
			<td>Trivial name: Mowiol 4-88; CAS number: 9002-89-5</td>
		</tr>
		<tr>
			<td>Puromycin dihydrochloride</td>
			<td>Sigma</td>
			<td>P8833</td>
			<td>From Streptomyces alboniger, reconstituted in sterile water&#160;</td>
		</tr>
		<tr>
			<td>Benchtop centrifuge, e.g. Rotina 380 R Benchtop centrigfuge</td>
			<td>Hettich Lab Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Sigma</td>
			<td>R0883</td>
			<td>Supplement with 10% (v/v) FBS and L-glutamine (2mM) to make up the optimal growth medium for 4T1 cells.</td>
		</tr>
		<tr>
			<td>SFCA Syringe filter 0.45 &#956;m</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes 10 mL</td>
			<td>BD Emerald</td>
			<td></td>
			<td>Disposable non-sterile syringes</td>
		</tr>
		<tr>
			<td>Tissue culture fluorescence microscope, e.g. EVOS-FL&#160;</td>
			<td>Life Technologies</td>
			<td></td>
			<td>Cell Imaging System equipped with a 10&#215; objective (PlanFL PH2, 10&#215;/0.25, &#8734;/1.2) and a colour camera</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution 10X&#160;</td>
			<td>Sigma</td>
			<td>59418C</td>
			<td>(0.5 % (w/v) trypsin, 0.2 % (w/v) EDTA) gamma irradiated by SER-TAIN process and without phenol red&#160;</td>
		</tr>
		<tr>
			<td>Wheat Germ Agglutinin Alexa Fluor 633 Conjugate&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>W21404</td>
			<td>Used at 1:1000 (2 &#181;g/mL) for cell immunofluorescence</td>
		</tr>
		<tr>
			<td colspan="4">Step 2) Establishment of in vivo tumor models.</td>
		</tr>
		<tr>
			<td>Digital caliper</td>
			<td>World Precision Instruments</td>
			<td>501601</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane 1000 mg/g</td>
			<td>Isocare</td>
			<td></td>
			<td>For inhalation</td>
		</tr>
		<tr>
			<td>Fluorescence Torch, e.g. NightSea Fluorescence Torch DFP-1</td>
			<td>Electron Microscopy Sciences</td>
			<td>SFA-LFS-RBS/GR</td>
			<td>Equipped with GFP and RFP emission filters and NightSea filter goggles (DFP-1)</td>
		</tr>
		<tr>
			<td>Syringes 0.3 mL U-100 insulin</td>
			<td>Terumo</td>
			<td></td>
			<td>29G &#215; 1/2&#39;&#39; - 0.33 &#215; 12 mm</td>
		</tr>
		<tr>
			<td>Standard materials/equipment for aseptic technique and animal maintenance</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Step 3) Production of [18F]BF4- using an automated radiotracer synthesis platform.</td>
		</tr>
		<tr>
			<td>15-crown-5</td>
			<td>Sigma-Aldrich</td>
			<td>188832</td>
			<td>CAS 33100-27-5</td>
		</tr>
		<tr>
			<td>Acetonitrile (anhydrous)</td>
			<td>Acros Organics</td>
			<td>326811000</td>
			<td></td>
		</tr>
		<tr>
			<td>Boron trifluoride diethyl etherate</td>
			<td>Sigma-Aldrich</td>
			<td>216607</td>
			<td>BF3.OEt2, purified by redistillation, &#8805;46.5 % BF3 basis. CAS 109-63-7</td>
		</tr>
		<tr>
			<td>Automated Radiotracer Synthesis (ARS) platform, e.g. FASTLab</td>
			<td>GE Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable cassettes for ARS platform, e.g. FASTLab cassettes</td>
			<td>GE Healthcare</td>
			<td></td>
			<td>FASTlab Developer pack</td>
		</tr>
		<tr>
			<td>Polygram Alox N/UV254 polyester sheets</td>
			<td>Macherey-Nagel</td>
			<td>802021</td>
			<td>RadioTLC plates, 40&#215;80 mm</td>
		</tr>
		<tr>
			<td>Strong anion exchange cartridge, e.g. Sep-Pak Accell Plus QMA Plus Light</td>
			<td>Waters</td>
			<td>WAT023525</td>
			<td>Condition with 1M NaCl (10 mL) and H2O (10 mL)</td>
		</tr>
		<tr>
			<td>Alumina neutral cartridge, e.g. Sep-Pak Alumina N Plus Light</td>
			<td>Waters</td>
			<td>WAT023561</td>
			<td>Condition with H2O (10 mL), acetone (10 mL) and air (20 mL)</td>
		</tr>
		<tr>
			<td>Water for injection USP</td>
			<td>GE Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen filter</td>
			<td>Millipore</td>
			<td>SE2M049I05</td>
			<td>Sterile 0.2 &#181;m FG Millex 13 mm</td>
		</tr>
		<tr>
			<td colspan="4">Step 4) In vivo imaging of NIS-FP expressing cells by nanoPET/CT.</td>
		</tr>
		<tr>
			<td>Isoflurane 1000 mg/g</td>
			<td>Isocare</td>
			<td></td>
			<td>For inhalation</td>
		</tr>
		<tr>
			<td>Preclinical PET/CT multimodal imaging instrument, e.g. nanoScan PET/CT</td>
			<td>Mediso Medical Imaging System, Budapest, Hungary</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Torch, e.g. NightSea Fluorescence Torch DFP-1</td>
			<td>Electron Microscopy Sciences</td>
			<td>SFA-LFS-RBS/GR</td>
			<td>Equipped with GFP and RFP emission filters and NightSea filter goggles (DFP-1)</td>
		</tr>
		<tr>
			<td>Rodent anesthesia induction chamber</td>
			<td>Vet-Tech</td>
			<td>AN010R</td>
			<td>With three-way valves (x2), tube mount connector for inlet, PVC tubing for gas inlet (2 m) and 22 mm scavenging tube (2 m)</td>
		</tr>
		<tr>
			<td>Rodent anesthesia system</td>
			<td>Vet-Tech</td>
			<td>AN001B</td>
			<td>Including animal face-mask suitably sized for animal of interest and isolflurane vaporizer</td>
		</tr>
		<tr>
			<td>Sterile physiological saline</td>
			<td>Thermo Scientific Oxoid</td>
			<td>BO0334B</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes 0.3 mL U-100 insulin</td>
			<td>Terumo</td>
			<td></td>
			<td>29G &#215; 1/2&#39;&#39; - 0.33 &#215; 12 mm, for intravenous injection of radiotracer</td>
		</tr>
		<tr>
			<td>Veterinary Scavenger</td>
			<td>Vet-Tech</td>
			<td>AN200</td>
			<td>VetScav filter weighing mechanism - 240 V with automatic temperature compensation and LED system</td>
		</tr>
		<tr>
			<td colspan="4">5) In vivo data analysis.</td>
		</tr>
		<tr>
			<td>Tera-Tomo Monte Carlo based full 3D iterative algorithm</td>
			<td>Mediso Medical Imaging System, Budapest, Hungary</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>VivoQuant Software</td>
			<td>Invicro LLC., Boston, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">6) Ex vivo analyses</td>
		</tr>
		<tr>
			<td>2-Methylbutane&#160;</td>
			<td>Sigma</td>
			<td>59070-1L-D</td>
			<td>Pre-cooled over liquid nitrogen to freeze OCT-embedded tissues</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>85040C</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slips 22&#215;50 mm</td>
			<td>VWR International</td>
			<td>SMITMCQ211022X50</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat, e.g. Cryostat MNT</td>
			<td>SLEE Medical</td>
			<td></td>
			<td>two-piece modular histology embedding machine equipped with an embedding module, a tissue storage compartment and a cold plate</td>
		</tr>
		<tr>
			<td>Cy5 AffiniPure Goat anti-Rabbit IgG (H+L)</td>
			<td>Jackson/Stratech</td>
			<td>111-175-144</td>
			<td>Used at 1:500 (2 &#181;g/mL)</td>
		</tr>
		<tr>
			<td>Dabco</td>
			<td>Sigma</td>
			<td>290734</td>
			<td>Stock 125 mg/mL</td>
		</tr>
		<tr>
			<td>Microtome blades, e.g. Feather S35&#160;</td>
			<td>CellPath</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Microscope (wide-field or confocal), e.g. Nikon Eclipse Ti-E Inverted Fluorescence Microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>Equipped with 10&#215;, 20&#215; (air) and ideally 40&#215; (oil) objectives and lasers/filters or filter cubes, respectively, that are suitable for Hoechst 33342, GFP and Cy5</td>
		</tr>
		<tr>
			<td>Automated Gamma Counter, e.g. 1282 Compugamma</td>
			<td>LKB Wallac Laboratory</td>
			<td></td>
			<td>99mTc-pertechnetate energy window 110-155 keV, 18F energy window 175-220 keV</td>
		</tr>
		<tr>
			<td>Hoechst 33342 solution</td>
			<td>Life Technologies</td>
			<td>H1399</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence adapter for dissecting microscope, e.g. NightSea Adapter</td>
			<td>Electron Microscopy Sciences</td>
			<td>SFA-LFS-RBS/GR</td>
			<td>Equipped with GFP and RFP emission filters</td>
		</tr>
		<tr>
			<td>O.C.T. compound&#160;</td>
			<td>VWR international</td>
			<td>361603E</td>
			<td></td>
		</tr>
		<tr>
			<td>Wax pen, e.g. PAP-PEN</td>
			<td>Dako UK Ltd</td>
			<td></td>
			<td>Wax pen to draw around tissue section to reduce required staining/washing solution volumes</td>
		</tr>
		<tr>
			<td>Paraformaldehyde solution 4 % (w/v) in PBS</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-281692</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-CD31</td>
			<td>Abcam</td>
			<td>ab28364</td>
			<td>Polyclonal anti-mouse used 1:50 (20 &#181;g/mL) for tissues immunofluorescence</td>
		</tr>
		<tr>
			<td>Microscope slides, e.g. Superfrost slides</td>
			<td>VWR, Lutterworth, UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-buffered saline (TBS)</td>
			<td>available from various suppliers.</td>
			<td></td>
			<td>Tris-buffered saline; 150 mM NaCl, 25 mM Tris/HCl at pH 7.4</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models at JoVE.com

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models (Video) | JoVE

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.

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 ...

Research

JoVE Journal

Cancer Research

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With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...

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Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features ...