Noninvasive imaging of immune responses

Significance Tumors are often surrounded and invaded by bone marrow-derived cells. Imaging the infiltration of such immune cells into tumors may therefore be an attractive means of detecting tumors or of tracking the response to anticancer therapy. We show that it is possible to detect these cells noninvasively by positron emission tomography (PET) via the surface markers displayed by them. The ability to monitor the immune response in the course of therapy will enable early determination of the efficacy of treatment and will inform decisions as to whether treatment should be stopped or continued. Noninvasive monitoring could therefore change how therapies are applied and assessed, to the benefit of many patients.

Two-Photon Imaging. Two-photon imaging was performed with an Olympus BX61 upright microscope (Olympus 25× 1.05 NA Plan Objective), fitted with a SpectraPysics MaiTai DeepSee laser. Images were acquired using 910-nm excitation and the following filters: second-harmonic emission (Collagen) (460-510 nm) and GFP (495-540 nm), separated by a 505-nm dichroic mirror, and a third filter (575-630 nm) for the Texas Red signal. Images were acquired with 5-μm Z resolution with Olympus FluoView FC1000 software. Tile images (Fig. 3H) were saved as JPEG files. Images in Fig. 2 F and G were processed to obtain a scale bar in Imaris, version 7.4.0; no intensity or contrast adjustments were made.
PET-CT Imaging. For all imaging experiments, mice were anesthetized using 1.5% isoflurane in O 2 at a flow rate of ∼1 L/min. Mice were imaged with PET-computed tomography (CT) using an Inveon small-animal scanner (Siemens). Each PET acquisition took ∼30 min. High-resolution Fourier rebinned PET images were reconstructed by a 3D ordered subsets expectation algorithm using maximum a priori (OSEM3D/MAP) with 18 MAP iterations and 2 OSEM3D iterations into 0.796 × 0.796 × 0.861 mm images on a 128 × 128 × 159 image matrix. Peak sensitivity of the Inveon accounts for 11.1% of positron emission, with a mean resolution of 1.65 mm. More than 100 counts were acquired per pixel, and the mean signal-to-noise ratio was greater than 20. CT images were acquired using an 80 kVp 500 mA X-ray tube over 360 projections on a 125-mm detector. A modified Feldkamp conebeam reconstruction algorithm (COBRA; Exxim) was used to reconstruct the CT images into a 110-μm isotropic image matrix of 512 × 512 × 768. Reconstruction of datasets, PET-CT registration, and image analysis were performed using IRW software (Siemens). Two-dimensional and 3D visualizations were produced using the DICOM viewer OsiriX (OsiriX Foundation).
Blood Half-Life Measurement of 18 F-VHHs. Mice were administered 30 ± 3 μCi of 18 F-VHH7 by i.v. tail-vein injection. Blood samples were obtained by retroorbital puncture using tared, heparinized capillary tubes. Blood samples and capillaries were weighed, and radioactivity was measured using a Perkin-Elmer Wallac Wizard 3′′ 1480 Automatic Gamma Counter. Values, expressed as percentages of the injected dose per gram of tissue, were fit (least squares) to a two-compartment biexponential decay model performed using GraphPad Prism 4.0c (Fig. S2).
Biodistribution Analysis of 18 F-or 64 Cu-VHHs. Mice were administered 296 ± 19 μCi of labeled VHHs by i.v. tail-vein injection. At 2 h postinjection, mice were euthanized, perfused with 1× PBS (20 mL), and dissected. Blood, urine, and tissues were excised, and their wet weight was determined. Tissue radioactivity was measured with a Perkin-Elmer Wallac Wizard 3′′ 1480 Automatic Gamma Counter. Statistical analysis was performed using GraphPad Prism 4.0c. Values are expressed as percentages of the injected dose (excretion subtracted) per gram of tissue.
PET Standard Uptake Value Calculation. Standard uptake value (SUV) is the derived ratio of tissue radioactivity concentration (Bq/mL) and the injected radioactivity per gram of the mouse's body weight. The calculation used the following equation: SUV = (region of interest radioactivity concentration)/(injected activity/ mouse total mass).
Analysis of the Purity of the Radiolabeled VHHs. The purity of the radiolabeled VHHs was assessed with TLC performed on silicaimpregnated glass sheets (ITLC plates; Pall Life Sciences). Plates for 18 F-VHHs were developed with 100% acetonitrile whereas 64 Cu-VHH plates were developed using 50 mM EDTA (pH 7.0) and analyzed using a Bioscan AR-2000 scanner operated by the WinScan V3 software package (Fig. S5).
Generation and Sequence Identity of VHH7 and VHHDC13. Two VHHs, VHH7 (anti-class II MHC) and VHHDC13 (anti-CD11b) were generated following standard procedures (2).    Cu-VHHs; inflammation around the injection site is clearly visible, attributable to influx of host-derived class II + or myeloid cells for D and F, respectively (arrows). Images are all window-leveled to the same intensity for better comparison.

Movie S5C
Movie S6A. Imaging the presence of tumor-associated class II MHC + cells using 18 F-VHH7 (anti-mouse class II MHC). A WT mouse was inoculated s.c. on the left shoulder with mouse B16 melanoma cells and imaged 7 d postinjection. Tumor cells lack mouse class II MHC molecules. Movies and images are representative of two to four mice with similar results.

Movie S6A
Movie S6B. Imaging the presence of tumor-associated class II MHC + cells using 18 F-VHH7 (anti-mouse class II MHC). A WT mouse was inoculated s.c. on the left shoulder with mouse B16 melanoma cells and imaged 7 d postinjection. Tumor cells lack mouse class II MHC molecules. Movies and images are representative of two to four mice with similar results.

Movie S6B
Movie S7. Imaging the presence of tumor cells using 18 F-FDG. A WT mouse was inoculated s.c. on the left shoulder with mouse B16 melanoma cells and imaged 7 d postinjection. Higher metabolic activity of the right shoulder's muscle relative to the left shoulder's muscle is probably due to the presence of the tumor on the left shoulder, which makes the mouse use its right shoulder more often than the left one to walk around. The tumor on the left shoulder is visible but with inferior specificity relative to VHHs. Movies and images are representative of two to four mice with similar results.

Movie S7
Movie S8A. Complete Freund's adjuvant (CFA) was injected into the left paw of C57BL/6 mice, and 18 F-VHHDC13 was used 24 h after CFA injection for imaging. PET-CT Images were obtained 1.5 h postinjection of 18 F-agent. Movies and images are representative of two to four mice with similar results.

Movie S8A
Movie S8B. Complete Freund's adjuvant (CFA) was injected into the left paw of C57BL/6 mice, and 18 F-VHHDC13 was used 24 h after CFA injection for imaging. PET-CT Images were obtained 1.5 h postinjection of 18 F-agent. Movies and images are representative of two to four mice with similar results.

Movie S8B
Movie S9. Complete Freund's adjuvant (CFA) was injected into the left paw of C57BL/6 mice, and 64 Cu-VHHDC13 was used 24 h after CFA injection for imaging. PET-CT images were obtained 4 h postinjection of 18 F-agent. Movies and images are representative of two to four mice with similar results.

Movie S9
Movie S10. Complete Freund's adjuvant (CFA) was injected into the left paw of C57BL/6 mice, and 18 F-VHH7 was used 24 h after CFA injection for imaging. PET-CT images were obtained 1.5 h postinjection of 18 F-agent. Movies and images are representative of two to four mice with similar results.
Movie S10 Movie S11. Complete Freund's adjuvant (CFA) was injected into the left paw of C57BL/6 mice, and 64 Cu-VHH7 was used 24 h after CFA injection for imaging. PET-CT Images were obtained 4 h postinjection of 64 Cu-agent. Movies and images are representative of two to four mice with similar results.
Movie S11 Movie S12A. Complete Freund's adjuvant (CFA) was injected into the left paw of C57BL/6 mice, and 18 F-FDG was used 24 h after CFA injection for imaging. PET-CT Images were obtained 1.5 h postinjection of 18 F-agent. Higher metabolic activity of the right shoulder's muscle relative to the left shoulder's muscle is probably due to the inflammation of the left paw, which makes the mouse use its right shoulder more than the left one to walk around. Movies and images are representative of two to four mice with similar results.
Movie S12A Movie S12B. Complete Freund's adjuvant (CFA) was injected into the left paw of C57BL/6 mice, and 18 F-FDG was used 24 h after CFA injection for imaging. PET-CT Images were obtained 1.5 h postinjection of 18 F-agent. Higher metabolic activity of the right shoulder's muscle relative to the left shoulder's muscle is probably due to the inflammation of the left paw, which makes the mouse use its right shoulder more than the left one to walk around. Movies and images are representative of two to four mice with similar results.
Movie S12B Movie S12C. Complete Freund's adjuvant (CFA) was injected into the left paw of C57BL/6 mice, and 18 F-FDG was used 24 h after CFA injection for imaging. PET-CT Images were obtained 1.5 h postinjection of 18 F-agent. Higher metabolic activity of the right shoulder's muscle relative to the left shoulder's muscle is probably due to the inflammation of the left paw, which makes the mouse use its right shoulder more than the left one to walk around. Movies and images are representative of two to four mice with similar results.
Movie S12C Image S2. Image of a lymph node of a WT mouse injected with 20 μg of VHH7-Texas Red 90 min before imaging. VHH7 (anti-mouse class II MHC) and VHHDC13 (anti-mouse CD11b) stain secondary lymphoid organs. VHHs were site-specifically labeled with Texas Red via sortagging. Images were acquired by two-photon microscopy. Movies and images are representative of two to four mice with similar results.

Image S2
Image S3. Image of a lymph node of an MHC-II −/− mouse injected with 20 μg of VHH7-Texas Red 90 min before imaging. VHH7 (anti-mouse class II MHC) and VHHDC13 (anti-mouse CD11b) stain secondary lymphoid organs. VHHs were site-specifically labeled with Texas Red via sortagging. Images were acquired by two-photon microscopy. Movies and images are representative of two to four mice with similar results.

Image S3
Image S1. Image of a lymph node of a WT mouse, with no VHH injection before imaging. VHH7 (anti-mouse class II MHC) and VHHDC13 (anti-mouse CD11b) stain secondary lymphoid organs. VHHs were site-specifically labeled with Texas Red via sortagging. Images were acquired by two-photon microscopy. Movies and images are representative of two to four mice with similar results. Image S1 Image S4. Image of a lymph node of a B6 mouse injected with 20 μg of DC13-Texas Red 90 min before imaging. VHH7 (anti-mouse class II MHC) and VHHDC13 (anti-mouse CD11b) stain secondary lymphoid organs. VHHs were site-specifically labeled with Texas Red via sortagging. Images were acquired by two-photon microscopy. Movies and images are representative of two to four mice with similar results. Image S4