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

This protocol meets all requirements set by United Kingdom (UK) legislation and the local Ethical Review panel. When following this protocol, ensure the procedures also meet all requirements dictated by national legislation and local Ethical Review panel. Ensure every experiment involving radioactivity is compliant with legislation and local rules and performed safely.

1. Engineering and characterization of cancer cells to express the radionuclide-fluorescence fusion reporter NIS-FP

NOTE: For simplicity, mEGFP A206K is abbreviated as "GFP", and mCherry as "RFP" in the subsequent sections of this protocol.

1. Generation of lentiviral particles
   1. To produce lentivirus particles, co-transfect 293T cells with the following four plasmids using a suitable transfection method: (i) the reporter gene encoding plasmid (pLNT SFFV NIS-GFP or pLNT SFFV NIS-RFP (see Supplementary Information), (ii) the third-generation lentiviral packaging plasmids pRRE and (iii) pRSV-Rev, and (iv) a virus envelope containing plasmid, e.g, pMD2.G. Pre-mix plasmids before adding them to the transfection mix. Perform transfection in a cell culture hood.
      NOTE: Additional transfection information is provided in Supplementary Information.
   2. Assess transfection success after 48 h by standard wide-field fluorescence microscopy with filter settings appropriate for the chosen fusion reporter (NIS-GFP or NIS-RFP).
      NOTE: Fluorescence signals are indicative of reporter gene transfection and therefore only a surrogate for successful co-transfection, not an indicator of successful virus production.
   3. Harvest the virus particle-containing supernatant using a syringe and remove floating cells and cell debris by filtering through a 0.45 µm sterile polyethersulfone (PES) filter. Transfer to a sterile 1.5 mL polypropylene reaction tube. Perform virus work in a cell culture hood and ensure no live virus leaves the contained environment.
2. Transduction and selection of NIS-FP expressing cancer cell lines
   1. Use pure fresh virus from step 1.1.3 mixed 1:1 (v/v) with the optimal growth medium of each cancer cell line (DMEM for MDA-MB-231 cells and RPMI 1640 for 4T1 cells; see Materials Table for media composition). Perform transduction in a cell culture hood.
      NOTE: A more general protocol is referred to in Supplementary Information.
   2. Transduce cancer cells with virus-containing medium in an incubator with humidified atmosphere containing 5% (v/v) CO₂ at 37 °C for 72 h (work on 6-well or 12-well scale using 1 mL or 0.4 mL of the virus mixture from step 1.2.1).
   3. Monitor target cell NIS-FP expression by fluorescence microscopy.
   4. Expand successfully transduced cells to a scale of 3-10 million cells using standard culture conditions (see ATCC for MDA-MB-231 and 4T1 cell lines).
   5. Use fluorescence-activated cell sorting (FACS) to purify NIS-FP expressing cells from non-transduced cells.
      NOTE: FACS can be a source of mycoplasma infections; it is recommended to check for mycoplasma before virus production and transduction but also after FACS and before step 1.3.
3. Characterization NIS-FP expressing cancer cell lines
   1. Confirm reporter expression by standard flow cytometry as described elsewhere²⁸.
   2. Confirm reporter integrity by standard immunoblotting as described elsewhere²⁹.
   3. Analyze intracellular fusion reporter localization by confocal fluorescence microscopy.
      NOTE: Staining with or co-expression of a plasma membrane marker⁶ and subsequent co-localization analysis³⁰ will facilitate this step.
   4. Analyze radiotracer uptake in NIS-FP expressing cell lines.
      NOTE: Any radioactive NIS substrate compatible with existing equipment is suitable, e.g. the iodide isotopes (¹²³I^–, ¹²⁴I^–, ¹²⁵I^–, ¹³¹I^–), ^99mTcO₄^–, ¹⁸⁸ReO₄^–, [¹⁸F]SO₃F^– or [¹⁸F]BF₄^–.
      1. Seed 10⁶ purified cells in 6-well plates in their optimal growth medium (see Table of Materials) one day prior to the experiment. Prepare all samples in triplicate and include control samples: (i) "specificity controls", i.e. NIS-FP expressing cells pre-incubated with a competitive NIS substrate to test uptake specificity; (ii) "parental cell controls", i.e. cells not expressing NIS-FP but receiving radiotracer to test basal uptake in parental cells.
      2. On the next morning, wash cells once with serum-free growth medium.
      3. Incubate the cells in serum-free growth medium in the presence of 50 kBq ^99mTcO₄^– or [¹⁸F]BF₄^– for 30 min at 37 °C (1 mL total volume). For specificity controls, pre-incubate the cells for 30 min with the competitive substrate NaClO₄^– (12.5 µM final concentration). Keep the competitive substrate concentration constant throughout the experiment.
      4. Collect the supernatant and transfer 100 µL to a prepared collection tube labeled "supernatant".
      5. Wash the cells twice with 1 mL ice-cold phosphate-buffered saline (PBS) containing Ca²⁺/Mg²⁺. Collect each wash solution and transfer 100 µL of each into a prepared collection tube labeled "wash1" or "wash2", respectively.
      6. Lift the cells by adding 500 µL PBS containing 0.25 % (w/v) trypsin and 0.53 mM EDTA, and incubating at 37 °C until the cells detach (check visually using a microscope). Transfer the suspension into a prepared collection tube labeled "cells". Wash the wells with 500 µL ice-cold PBS containing Ca²⁺/Mg²⁺ and add to the tube "cells". Pellet the cells by centrifugation (250 x g, 4 min, 4 °C).
      7. Count all four sample types from each well using a gamma counter appropriately set for the radioisotope of choice (here: ^99mTc or ¹⁸F).
         NOTE: Due to the large number of samples in this assay, it is recommended to use an automated γ-counter capable of automatic decay correction.
      8. Analyze data by summing obtained gamma counts of samples from each well to determine a total radioactivity count per well.
         NOTE: take aliquoting in steps 1.3.4.4 and 1.3.4.5 into account by multiplying numbers by 10 for "supernatant", and "wash" fractions.
      9. Express cellular radiotracer uptake as %Uptake as indicated in Equ.1. Calculate averages and standard deviations from triplicate experiments.
                   (Equ.1)
         NOTE: Here, reporter validation experiments are described, but non-reporter-related cellular functions (e.g. proliferation, cancer cell invasion, gene expression etc.) are ultimately application-specific and the responsibility of each user.

2. Establishment of in vivo tumor models

1. Use only fully characterized and validated cells for in vivo experiments. Check fluorescence of cells prior to administration by any suitable technique (e.g. fluorescence microscopy, flow cytometry) on every occasion.
2. Establish the tumor model in 5-6 weeks old young-adult female mice. Use BALB/cAnNCrl or BALB/cAnN.Cg-Foxn1^nu (BALB/c Nude) mice for the 4T1 tumor model and NOD.Cg-Prkdc^scid Il2rg^tm1WjI/SzJ (NSG) mice for the MDA-MB-231-based tumor model. Shave animals locally and use aseptic technique. Directly inject 50 µL of a suspension containing 10⁶ NIS-FP expressing cancer cells/mL into the mammary fat pad between the fourth and the fifth nipple³¹.
   NOTE: To improve injection accuracy, it is recommended performing the injection under general anesthesia using an inhalable anesthetic such as isoflurane (1-2 % (v/v) in O₂).
   NOTE: Surgical implantation of tumor pieces from NIS-FP expressing tumors is an alternative approach to tumor model establishment³¹.
3. Check fluorescence of cells at injection sites after administration and in the early days post injection. Use a fluorescence torch and filter glasses suitable for the FP of choice.
4. Monitor tumor growth and check for any clinical signs (particularly at later time points).
   NOTE: For superficial tumors, i.e. the orthotopic breast tumors in this protocol, use calipers and the formula for the mean tumor diameter (MTD) = ½·(L+W)³².

3. Production of [¹⁸ F]BF ₄ ^– using an automated radiotracer synthesis (ARS) platform.

NOTE: Here, the automated [¹⁸F]BF₄^– synthesis based on the method of ¹⁸F addition to boron trifluoride is described. Users of a more widely available ARS platform (see Table of Materials), can download the corresponding Extensible Markup Language (XML) file required to run the automated sequence on this platform (Supplementary File). A detailed explanation of the cassette layout shown in Figure 1 is provided in (Table 1) as well as a detailed description of each step in the XML sequence file (Table 2) to support translation to any other automated platform.

1. Set up the ARS platform as described in Table 1 and ensure it is operational with the correct XML file loaded onto the control computer. Ensure the ARS platform is placed in a chemical hood suitable for safe work with GBq amounts of radioactivity.
2. Aspirate the cyclotron-produced [¹⁸F]F^– (typically 1.5-2 GBq in 2-7 mL [¹⁸O]H₂O) via the inlet reservoir (V6).
3. Trap the radioactivity on an anion exchange resin (e.g. quaternary ammonium anion exchange cartridge; V5 to V4), recover the [¹⁸O]H₂O in a separate vial (V1).
4. Elute the [¹⁸F]F^– (reverse elution, V5 to V4) into the reactor (V8) with 750 µL of 0.9 % (w/v) saline solution (V2), followed by 1.5 mL of acetonitrile (V16).
5. Remove the water by azeotropic evaporation under vacuum and nitrogen flow by heating at 105 °C then 120 °C for 5 min.
6. Reduce the reactor temperature to 80 °C.
7. Add 800 µL of 15-crown-5 in anhydrous acetonitrile (46 mg, 0.21 mmol) to the dry [¹⁸F]F^– through the central port of the reactor (V8).
8. Add 850 µL of BF₃.OEt₂ in anhydrous acetonitrile (0.16 mg, 1.13 µmol) to the reactor.
9. React for 5 min while returning to room temperature.
10. Pass the reaction mixture through an aluminum oxide cartridge (V17 to V18) to trap the trifluoroborate.
11. Return the reaction mixture to syringe S2 and dilute with water (approx. 1.6 mL).
12. Pass the reaction mixture through a second anion exchange cartridge (V19 to V20) to trap the [¹⁸F]BF₄^– product.
13. Wash the reactor with water (approx. 5.5 mL).
14. Pass the resulting solution through the alumina and second anion exchange cartridges.
15. Rinse syringes S2 and S3 with water.
16. Wash the second anion exchange cartridge with water and dry it with nitrogen gas.
17. Elute the product (V19 to V20) with 1 mL of 0.9 % NaCl (V14) into syringe S3.
18. Transfer the product (400-500 µL) via the outlet line (V21) to a 1 mL glass collection vial.
    NOTE: Molar activity is an important aspect of every radiotracer. However, its routine determination is not only time-consuming but also requires significant amounts of the freshly synthesized [¹⁸F]BF₄^–, such that it becomes a limiting factor for the number of animals that can be imaged. To test reproducibility and molar activities of the resultant [¹⁸F]BF₄^–, it is recommended to schedule a few dedicated test-runs for this purpose. For more details about molar activities, please refer to the Discussion section.

4. In vivo imaging of NIS-FP expressing cells by nanoPET/CT

1. Animal preparation
   1. Anesthetize mice with 1.5-2.0 % (v/v) isoflurane in O₂ at a flow rate of 1.0-1.5 L/min in an induction chamber. To check for sufficient anesthesia look for absence of the pedal reflex.
   2. Ensure to apply vet ointment on animal eyes to prevent dryness while under anesthesia
   3. Move the mouse onto a heating pad with the nose in an anesthetic supply mask and warm the tail (e.g. by dipping it into 37 °C water or using an infrared light lamp).
   4. Dilute the freshly prepared sterile-filtered [¹⁸F]BF₄^– solution to 5 MBq per 50 µL with 0.9 % sterile saline.
   5. Using a syringe connected to a hypodermic needle (gauge 29-31), draw 100 µL of the [¹⁸F]BF₄^– solution, measure the radioactivity in the syringe, and note the value and the time of the measurement.
   6. Intravenously administer 50 µL of the [¹⁸F]BF₄^– solution into the pre-warmed tail vein.
   7. Measure the remaining radioactivity in the syringe and note the value and the time of the measurement. The difference between values measured at steps 4.1.7 and 4.1.5 is the injected dose (ID).
   8. Set a timer to count down from 45 min (start time of PET imaging = 0 min).
   9. Place the mouse onto the bed of the nanoPET/CT scanner and ensure the anesthetic supply is correctly re-attached.
   10. Check anesthesia remains complete by testing for absence of the pedal reflex.
   11. Ensure the mouse is positioned on the bed in the desired way, e.g. the 'sphinx'-like position.
   12. Install animal monitoring devices according to the manufacturer's recommendations, e.g. a rectal temperature probe, a probe measuring animal breathing, or electrodes for recording electrocardiograms. Check the proper function of all instruments.
       NOTE: For in vivo specificity tests with the NIS radiotracer [¹⁸F]BF₄^–, animals are imaged as described above, and then rested awake until the radioactivity has decayed sufficiently to be regarded as negligible, e.g. 48 h later when only 1.3·10⁻⁶ % residual ¹⁸F radioactivity will be present in the animal. In the subsequent imaging session, the competitive substrate ClO₄^– is administered at a dose of 200 mg/kg 30 min prior to radiotracer administration, and imaging is performed as described above.
2. Imaging by nanoPET/CT
   1. Set the desired CT imaging parameters, e.g. using the nanoPET/CT 55 kVp tube voltage, set the exposure time to 1200 ms with one-degree angular stepping and 180-degree projections.
   2. Set the parameters for PET image acquisition. Use static scan PET parameters with a duration of 30 min, 1:5 coincidence mode and 400-600 keVp energy window.
   3. At countdown time = 15 min start CT image acquisition.
   4. At countdown time = 0 min start PET image acquisition.
      1. If serial animal imaging is required, let animals fully recover from anesthesia, i.e. regain consciousness under supervision. Subsequently, transfer them to a maintenance unit.
      2. If this is the terminal imaging session, proceed to animal euthanasia by either anesthetic overdose, rising concentration of carbon dioxide, or dislocation of the neck.

5. In vivo data analysis

1. Reconstruct the PET/CT data using a Monte Carlo-based full 3D iterative algorithm. Ensure that corrections for attenuation, dead time, and radioisotope decay are considered. For details refer to the manufacturer's instructions of the PET/CT instrument in use.
2. Check CT and PET images are correctly co-registered and save the data in a suitable exchange format such as 'Digital Imaging and Communications in Medicine' (DICOM).
3. Analyze images
   1. Load the reconstructed DICOM files into a suitable image analysis software that enables the recognition and delineation of regions of interest (ROIs) and subsequent PET signal quantification in these ROIs.
   2. Segment the ROIs using manual or adaptive thresholding to define ROIs^33,34 using a suitable software package. Anatomical image information from the CT scan helps guide ROI assignment, e.g. superficial tumors or lung volumes.
   3. Use the analysis software as per manufacturer's instructions and ensure data are calibrated to the injected radioactivity dose and corrected for attenuation and radioactive decay.
4. Draw graphs showing data from this in vivo quantification. Express data as either percent injected dose/volume (%ID/mL) or standard uptake value (SUV), which is an alternative measure considering the radioactivity in the whole body of the subject.
   1. Calculate %ID/g values assuming the tissue density to be like water, i.e. ~1 g/L. It is noteworthy that this assumption can be invalid for organs with significantly different densities, such as lung or bone.
   2. Calculate different SUVs to estimate the true SUV (e.g. SUVmean, SUVmax); SUVmax is more reliable for small objects and is more frequently used than SUVmean³⁵.

6. Ex vivo analyses

Perform the listed downstream analyses: (i) fluorescence imaging of organs containing fluorescent cancer cells (primary tumor and metastases) during animal dissection, (ii) measurement of radiotracer tissue distribution, and (iii) histologic or (iv) cytometric assessment of cancerous organs.

1. Measurement of radiotracer distribution by γ-counting (ex vivo biodistribution) and ex vivo fluorescence imaging of cancerous tissues.
   1. Measure radioactivity of the whole dead animal and note the value and the time.
   2. Dissect the animals and harvest the following tissues: lung, heart, blood (using 20 mm glass capillaries), liver, stomach, kidneys, spleen, small and large intestines, thyroid and salivary glands, a piece of muscle from the leg, and bone of the rear femurs, and relevant and dissectible lymph nodes and cancerous tissues.
   3. Measure the radioactivity of the remaining carcass first including then excluding the tail and note the values and the times of measurement.
      NOTE: Radioactivity in the tail can be considered stemming from radiotracer that was mis-injected and thus did not reach circulation; hence, this amount of radiotracer was not contributing to the injected dose. The tail radioactivity serves also as a retrospective parameter of injection quality.
   4. Weigh all tissues (use pre-weighed tubes).
   5. Take photographs of cancerous organs in daylight and under fluorescence light.
      NOTE: Use a camera stand to keep the distance between camera lens and organ constant (or use a dedicated commercial instrument for this purpose).
   6. Embed organs/tissues intended for downstream histology into OCT or immerse them in formalin for fixation. For other downstream applications sample preparation can differ.
   7. Prepare radiotracer calibration standards in duplicate, e.g. 0 to 1000 kBq [¹⁸F]BF₄^–.
      NOTE: Calibration standards are required to (i) relate the measured counts per min to radioactivity values (in kBq), and (ii) simplify decay correction; ¹⁸F^– can replace [¹⁸F]BF₄^–.
   8. Count the radioactivity of all harvested tissues using a γ-counter together with radioactivity calibration standards from step 6.1.7. Note the time of measurement. If count rates are too high (i.e. outside of linearity of calibration standard or indicated by too high detector dead times), re-count the samples two radiotracer half-lives later.
   9. Present data either as %ID/g or standard uptake values (SUV) (Equ.2).
      SUV =        (Equ.2)
   10. Discard all harvested tissues that are not required for further downstream analyses according to local waste management rules.
2. Analyze cancerous tissues by cytometry or histology according to the user's preferences and standard protocols (as described elsewhere^3,6,28).