Combination Fluorescence Bioluminescence Imaging with Spectral

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Combination Fluorescence Bioluminescence Imaging with Spectral Powered By Docstoc
					           Combination Fluorescence/Bioluminescence Imaging
        with Spectral Separations to Noninvasively Monitor Tumor
         Growth and Angiogenesis in Two and Three Dimensions
                                                              Ning Zhang1, Chaincy Kuo1, Ed Lim1, Scott Lyons2, Heng Xu1, Meng Yang3,
                                                            Robert M. Hoffman3, Peter Lassota1, Mark Roskey1, Brad Rice1, Kevin P. Francis1
                                                                             Caliper Life Sciences, Hopkinton, MA USA
                        Dept of Molecular Imaging, Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 0RE, United Kingdom
                                                                 AntiCancer, Inc., 7917 Ostrow St., San Diego, California, 92111, USA

   Abstract                                                                                                                              Results (cont)
     Bioluminescent and fluorescent labeled tumor cells have added new dimensions for tumor detection                                                          GFP Tumor           GFP Tumor                   RFP Tumor              RFP Tumor
     through optical imaging. Application of optical imaging in preclinical settings has facilitated                                                              s.c.              Prostate                      s.c.                  Brain
     development and validation of new therapeutic agents. We have used various tumor models for
     determining the sensitivity of detection of fluorescent protein labeled tumors with optical imaging. We
     demonstrated that fluorescent protein labeled tumors can be readily detected in a subcutaneous
     model, an orthotopic prostate tumor model and an orthotopic brain tumor model using the IVIS®
     Spectrum®. Spectral imaging of the tumors with combinatory filter sets allows the separation of
     fluorescent protein mediated tumor signal from auto-fluorescence background through “unmixing”, a
     novel feature of the Living Image® software. The unmixing process can significantly boost signal to
     background ratio, and thus the sensitivity of tumor detection.
     Furthermore, spectral imaging was able to detect and differentiate signals of two fluorescence proteins
                                                                                                                                                                     Ex 465 nm         Ex 465 nm                   Ex 535 nm               Ex 535 nm
     based on the difference of their spectral properties. We also performed 3D fluorescent imaging                                                  Tumor           Em 520 nm         Em 520 nm                   Em 600 nm               Em 600 nm

     tomography (FLIT) analysis of tumors and were able to add tumor depth measurement to the standard                                               volume:         25   mm3          421   mm3                  21   mm3               17 mm3
     2D signal quantification. We finally applied dual bioluminescent/fluorescent imaging to monitor tumor                                                                Figure 3. Fluorescence Imaging of GFP and RFP Tumors
     development as well as tumor induced angiogenesis activity in VEGFR2-luc knock-in mice. The
     processes of tumor development and angiogenesis were dissected with different imaging modalities.
                                                                                                                                                       LL2-GFP                             LL2-RFP                                       LL2-Luc
     The data demonstrate that the combination of fluorescence and bioluminescence offers many                                                   In vivo        Ex vivo               In vivo          Ex vivo                       Dorsal            Ventral
     advantages for optical imaging.


      Spectral Epi-illumination
                                                            Image 5-11
              Ex    Em    Signal      BKG        Ratio
                                                                                                                                                     Ex 465, Em 520                        Ex 535, Em 600
         1    465   520   3.1E-04    3.8E-05      8.3
         2    465   540   1.3E-04    3.3E-05      3.9                                                                                        Figure 4. Orthotopic Lung Tumor Detection - Five million LL2 tumor cells were injected intratracheally to Nu/Nu mice
         3    465   560   6.8E-05    2.7E-05      2.5                                                                                        and imaged on day 12.
         4    465   580   4.0E-05    2.2E-05      1.8
         5    465   600   3.3E-05    2.0E-05      1.7
         6    500   540   2.2E-04    1.4E-05     15.3
                                                                                                                                                  Ex 500 nm / Em 540 nm                                                        FLIT Dorsal
         7    500   560   1.1E-04    1.4E-05      7.5
         8    500   580   5.7E-05    1.4E-05      4.2
         9    500   600   4.8E-05    1.3E-05      3.6
        10    500   620   3.1E-05    1.2E-05      2.5
        11    500   640   2.0E-05    1.1E-05      1.9
        12    500   660   1.3E-05    8.1E-06      1.6
              Unmixing    2.1E-04    1.9E-06    106.5

                    Tumor volume: 25    mm3

                                            Unmixing: Signal/BKG 106

                                                                                                                                                                                                         Dorsal View                               Left Lateral View
                                                                                                                                                                                                               10% of max Fluorescent light source displayed

                                                                                                                                                 Figure 5. 3D-Reconstruction of PC3-GFP Tumor following Fluorescence Imaging - A nu/nu mouse with
                                                                                                                                                 orthotopic prostate tumor was imaged through trans-illumination. The imaged were applied to 3D re-construction.

                                                                                                                                                     Fluorescence Imaging                                       Bioluminescence Imaging

     Figure 1. Fluorescence Imaging of s.c. PC3-GFP Tumor - PC3-GFP tumor of 1 mm3 was implanted s.c. to the right flank of a
     nu/nu mouse. On day 18, the mouse was imaged. Unmixing of the fluorescence signal of the PC3-GFP tumor from the auto-fluorescence
     background using the Living Image software was demonstrated.

                                                                                                                                                             GFP signal                                      Tumor induced VEGFR2-luc expression
                                                                                                                                                    Figure 6. Monitoring of Tumor Development and Angiogenesis with Optical Imaging -
                                                                                                                                                    VEGFR2-luc KI mice were implanted with 1 million LL/2-GFP tumor cells and imaged at day 18

                                                                                                                                         Materials and Methods
                                                                                                                        x10-3             Bioluminescence imaging
                                                                                                                                          In vivo imaging of luciferase activity from the spontaneous pancreatic tumor model was performed using an
                                                                                                                                          IVIS Imaging System. Mice were injected i.p. with 150 mg/kg of luciferin and imaged after 10 minutes in three
                                                                                                                                          positions (ventral, left flank and right flank). Photons emitted from the pancreas region in each position were
                                                                                                                                          quantified using Living Image software and the sum of these measurements was used as the total biolumi-
                                                                                                                                          nescence signal from the pancreas.
                                                                                                                                          Fluorescent imaging of xenografts
                                                                                                                                          Xenografts were established in female nu/nu mice. In vivo fluorescent imaging was accomplished by using an
                                                                                                                 Min = 2.27e-5
                                                                                                                 Max = 9.28e-4
                                                                                                                                          IVIS Spectrum. Fluorescent signals from specific regions were quantified using Living Image software.

                                    Sig/BKG: 3.2                             Sig/BKG: 37
                                                                                                                                         The ability to monitor both bioluminescent and fluorescent signals non-invasively in vivo using one
                                                                                                                                         imaging platform has tremendous advantages to the user. The IVIS Spectrum is a state-of-the-art
                                                                                                                                         optical imaging platform that can quantitatively 3-dimentional reconstruct optical signals from deep
                                                                                                                                         within small animals, regardless of whether the optical reporter is a luciferase, a fluorescent protein or
                                                                                                                                         a fluorochrome. In this study, we were able to detect both GFP and RFP labeled tumors in vivo. By
                                                                                                                                         performing spectral unmixing with the Living Image software, we were able to accurately separate the
                                                                                                                                         fluorescence signal from the auto-fluorescence, to dramatically increase the sensitivity of detection of
                                                                                                                                         the fluorescently labeled tumors. Moreover, by implanting a GFP labeled LL2 tumor in a VEGFR2-luc
                                                                                                                                         expressing transgenic knock-in mouse, we were able to visualize both growth of the cancer cell line
                                                                                                                                         (fluorescence) and angiogenesis (bioluminescence), simultaneously.
                                         Ex 465                                    Ex 570
                                         Em 520                                    Em 620
                                                        Tumor volume: 394 mm3

      Figure 2. Fluorescence Imaging of Dual-Labeled HT1080 GFP/RFP Tumor - Dual colored HT1080 tumor of 1 mm3 was
      implanted to the tibia (orthotopic) of a nu/nu mouse. On day 18, the mouse was imaged.