ANALYTICAL SCIENCES 2001, VOL.17 SUPPLEMENT i531 2001 © The Japan Society for Analytical Chemistry

Synchrotron Radiation Microangiography for Real-Time Observation of Angiogenic Vessels in Cancer

Keiji UMETANI,1† Takenori YAMASHITA,2 Nobunao MAEHARA,2 Shigeki IMAI,2 and Yasumasa KAJIHARA2

1† Life & Environment Division, Japan Synchrotron Radiation Research Institute, SPring-8, Mikazuki, Sayo, Hyogo 679-5198, Japan (E-mail: [email protected]) 2 Department of Diagnostic Radiology, Kawasaki Medical School, Matsushima, Kurashiki, Okayama 701-0192, Japan

Microangiography experiments were carried out for depiction of angiogenic vessels in a rabbit model of cancer using a direct-conversion type detector and a third generation synchrotron radiation source at the SPring-8. An image detector with 10-11 µm spatial resolution has been developed for real-time small-field imaging. The detector incorporates an X-ray SATICON pickup tube with a beryllium faceplate for X-ray incidence to a photoconductive layer. In synchrotron radiation radiography, a long source-to-object distance and a small source spot can produce high-resolution images. The VX2 cancer cells had been transplanted into the rabbit auricle. At one, three, and seven days after transplantation, microangiographic images were obtained at a monochromatic X-ray energy of 33.2 keV just above the iodine K-edge energy. Small tumor blood vessels with diameters of 20-30 µm in an immature vascular network were visualized after contrast material injection into the auricular artery.

(Received on August 8, 2001; Accepted on October 5, 2001)

A photon beam generated by bending the path of electrons at carried out for depiction of angiogenic vessels in a rabbit model relativistic speeds is called synchrotron radiation, which is of cancer at the monochromatic X-ray energy of 33.2 keV just characterized by a small source spot and a radiation beam of above the iodine K-edge energy. Sequential images of tumor very high spectral brightness. Medical imaging using blood vessels induced by transplanted VX2 cancer cells in rabbit synchrotron radiation has been investigated since dual-energy auricles were obtained after iodine contrast material injection subtraction coronary angiography was started at Stanford into the auricular artery. Tumor-induced small blood vessels University and the Stanford Synchrotron Radiation Laboratory in with 20-30 µm diameters were visualized. In addition, two sets 1979.1,2 Difference in X-ray absorption by iodine contrast of sequential images were acquired at different rotation angles for material at energies just above and just below the iodine K-edge stereoscopic observation of complex vascular networks. energy has been used for high-sensitivity imaging of a diluted Methods contrast material. Research groups at several synchrotron radiation facilities have improved dual-energy imaging systems The X-ray direct-conversion type SATICON tube incorporates for human studies.3-5 a photoconductive target layer of amorphous selenium. In Japan, the first human study of intravenous coronary Absorbed X-rays in the photoconductive layer of the tube are angiography was performed using a single-energy approach at directly converted into electron-hole pairs and signal charges are the Accumulation Ring of the High Energy Accelerator Research read out by electron beam scanning. Organization in May 1996.6 In this approach, the monochromatic The X-ray direct-conversion type pickup tube was developed X-ray beam at energies only above the iodine K-edge energy in the 1960’s using a PbO photoconductive layer; and the image was employed to produce the highest contrast image of the detector was combined with a high power X-ray generator for iodine contrast material. The single-energy approach has also observation of real-time topography images in material science been applied to intra-arterial coronary microangiography.7 In fields. Photoconductive materials were improved for high- addition to coronary angiography, this intra-arterial resolution imaging. In place of the PbO photoconductive layer, microangiography system has been investigated as a diagnostic the X-ray SATICON tube with an amorphous photoconductive tool for circulatory disorders and early stage malignant tumors.8,9 Se-As alloy target was developed for use in synchrotron A conventional angiography system incorporating an X-ray radiation experiments of live topography at the Photon Factory.12 image intensifier and a video camera is not intended for The direct-conversion camera was much improved through detection of small blood vessels having diameters of 200 µm or introduction of a high-definition television (HDTV) system less because it is designed for large-field digital angiographic which increased resolution to 1050 scanning lines. The new X- imaging with a 1024×1024-pixel format. ray SATICON camera for medical imaging has identical At the SPring-8, the digital microangiography system with a resolution of 1050 scanning lines. The new camera, however, spatial resolution down to 10-11 µm has been developed using a can take digital images at a maximum speed of 30 images/s. direct-conversion type detector incorporating the X-ray Sequential images were obtained with an input field of view SATICON pickup tube.10,11 Microangiography experiments were of 9.5 mm×9.5 mm.10,11 Digital images were stored in a digital-

i532 ANALYTICAL SCIENCES 2001, VOL.17 SUPPLEMENT

STORAGE RING &

EXPERIMENTAL HALL BIOMEDICAL IMAGING CENTER

BL20B2 BEAMLINE

Fig. 1. An overview of the medium length bending-magnet BL20B2 beamline at the SPring-8. The X-ray beam produced by the bending magnet passes out of the Experimental Hall surrounding the storage ring and enters the Biomedical Imaging Center.

8GeV ELECTRON BEAM

MONOCHROMATIC X-RAY BENDING MAGNET DOUBLE-CRYSTAL MONOCHROMATOR X-RAY

SYNCHROTRON SLITS

RADIATION

OBJECT

X-RAY SATICON Fig. 2. An illustration of an experimental arrangement for microangiography and a photograph of the X-ray SATICON camera. image acquisition system after analog-to-digital conversion, bandwidth are used for imaging. In Fig. 2, the storage ring and synchronizing timing with electron beam scanning in the pickup the beamline are high-vacuum tubes. The monochromatic X-ray tube. An equivalent pixel size projected onto the input window comes out from the vacuum tube into the atmosphere by passing was 9.5 µm in the case of a 1024×1024-pixel format. through a beryllium window that is located down stream of slits. X-rays transmitted through the object are detected by the Experimental imaging detector. Coronary angiography studies using the monochromatic X-ray Experiments were performed in the Biomedical Imaging have been performed mainly at a second generation of Center. Distance between the source point in the bending magnet synchrotron radiation facilities.3,4 A first generation of and the detector was about 210 meters. A fan-shaped beam with synchrotrons was used primarily for particle physics; and any a cross-section size of 300 mm width by 20 mm height is research done with synchrotron radiation was a parasitic activity. produced in the Biomedical Imaging Center. Vertical divergence The second generation of synchrotron radiation source is of the X-ray beam was 9 µrad. The beam size, however, was dedicated to production of synchrotron radiation providing a adjusted to 9.5 mm×9.5 mm for small-field imaging by slits. steady source of X-rays for research. The nearly parallel X-ray beam was used for imaging without The third generation of synchrotron radiation is characterized geometrical unsharpness because of the small X-ray source size by the small source spot and radiation beams of very high and the very long source-to-object distance. spectral brightness. There are only three hard X-ray third The storage ring was operated at 8 GeV electron beam energy generation synchrotron radiation facilities. The SPring-8 opened and beam current was 80-100 mA. The energy of in 1997 to join the European Synchrotron Radiation Facility monochromatic X-rays was adjusted to 33.2 keV, just above the (ESRF) in Grenoble and the Advanced Photon Source (APS) in iodine K-edge energy, to produce the highest contrast image of Illinois.13 iodine contrast material. X-ray flux at the object location was The medium length bending-magnet beamline BL20B2 at the 5×107 photons/mm2/s at an electron beam current of 100 mA. SPring-8 shown in Fig. 1 is dedicated mainly to basic medical Results and Discussion science using biological specimens and small animals.10 The experimental arrangement for microangiographic imaging and Performance of the direct-conversion detector was evaluated the photograph of the X-ray direct-conversion type SATICON by taking an image of a specially ordered gold bar chart. camera are shown in Fig. 2. Thickness of the gold chart was 23 µm, and pattern widths were Synchrotron radiation has a broad and continuous spectrum 8.8-31.4 µm. Digital images were acquired with the 1024×1024- from the infrared to the X-ray region. A fixed-exit double crystal pixel format. The chart image was obtained with the input field monochromator employing Si <311> crystals selects a single of view of 9.5 mm×9.5 mm. In the chart image, stripes of 11.8 synchrotron radiation energy; and X-rays with a small energy µm in width were readily visible, but 8.8-µm stripes did not

ANALYTICAL SCIENCES 2001, VOL.17 SUPPLEMENT i533

appear because pixel size was 9.5 µm. Limiting spatial blood vessels are an intrinsic part of tumor development and resolution was 10-11 µm and nearly equal to 9.5 µm, which is progression. The tumor growth rate is slow before blood vessel equivalent to Nyquist frequency. Resolution was about 20 times formation and rapid after vessel formation.14,15 Radiographic higher than that of conventional systems. depiction of tumor-induced small vessels that feed a space- A tumor stimulates growth of small blood vessels for feeding occupying lesion is a useful tool for diagnosis of malignant the tumor itself. It has been proposed that tumor-induced small tumors.

L1 0.1mm R1

L2 R2

L3 R3

L4 R4

Fig. 3. A pair of consecutive images for stereoscopic observation obtained at three days after transplantation of the VX2 cancer cells into the rabbit auricle.

i534 ANALYTICAL SCIENCES 2001, VOL.17 SUPPLEMENT

We conducted an experimental study using rabbit VX2 vascular networks. The synchrotron radiation imaging system is carcinoma transplanted into rabbit auricles to evaluate growth of a useful tool for evaluating malignant tumor tumor-induced small blood vessels. A VX2 carcinoma is a microangioarchitecture. rapidly growing solid tumor. Tumor cells are easily transplanted Acknowledgments into other rabbits. Japanese white rabbits weighing about 3 kg were used for microangiographic imaging. Rabbits were The authors wish to thank Mr. Tadaaki Hirai, Mr. Toshiaki anesthetized by intravenous injection of pentobarbital sodium. Kawai, and Mr. Katsuhiko Suzuki of Hamamatsu Photonics K.K. The VX2 cancer cells were subcutaneously injected into the for development of the X-ray SATICON tube. The authors also rabbit auricle. thank Mr. Sadao Takahashi of Hitachi Denshi Techno-System, Microangiography experiments were performed at one, three, Ltd. for providing the camera system. and seven days after transplantation. Tumor volume increased sequentially, and diameter and thickness of the tumor were about References 10 mm and 5 mm at seven days, respectively. Rabbits were 1. R. Lewis, Phys. Med. Biol., 1997, 42, 1213. similarly anesthetized. A 24 gauge intracatheter was inserted into 2. E. Rubenstein, E. B. Hughes, L. E. Campbell, R. Hofstadter, the auricular artery. Volume of the contrast material was 2.4 ml R. L. Kirk, T. J. Krolicki, J. P. Stone, S. Wilson, H. D. per one injection at a speed of 0.2 ml/s. Contrast material was Zeman, W. R. Brody, A. Macovski, and A. C. Thompson, injected into the auricular artery by a mechanical power injector; Proc. SPIE, 1981, 314, 42. and timing of the injection was controlled by the digital-image 3. E. Rubenstein, G. S. Brown, D. Chapman, R. F. Garrett, J. acquisition system. C. Giacomini, N. Gmur, H. J. Gordon, W. M. Lavender, J. Microangiographic images were obtained sequentially at a Morrison, W. Thomlinson, A. C. Thompson, and H. speed of 5 images/s. Exposure time and X-ray dose per image Zeman, in Synchrotron Radiation in the Biosciences, ed. B. were 0.2 s and 0.6 mGy, respectively. Detection efficiency of the Chance et al., Oxford University Press, New York, 1994, detector was 14 % at an energy of 33.2 keV. Sequential images 639. were stored in the digital-image acquisition system with 10-bit 4. T. Dill, W. -R. Dix, C. W. Hamm, M. Jung, W. Kupper, M. resolution and 1024×1024-pixel format. The total number of Lohmann, B. Reime, and R. Ventura, Synchrotron acquired frames was 50, and total imaging time was 10 s. All of Radiation News, 1998, 11(2), 12. our experiments on animals conformed to the SPring-8 Guide for 5. H. Elleaume, S. Fiedler, F. Estève, B. Bertrand, A. M. Care and Use of Laboratory Animals. Charvet, A. Bravin, P. Berkvens, G. Berruyer, T. Brochard, Representative images from two microangiographic sequences Y. Dabin, A. Draperi, G. Goujon, G. Le Duc, C. Nemoz, M. are shown in Fig. 3. Sequential images of the transplanted tumor Perez, M. Renier, P. Suortti, W. Thomlinson, and J. F. Le were taken at three days after transplantation by the direct- Bas, ESRF Highlights 2000, 2001,11. conversion detector. In Fig. 3, a pair of consecutive images (L1- 6. S. Ohtsuka, Y. Sugishita, T. Takeda, Y. Itai, J. Tada, K. L4 and R1-R4) is a stereoscopic pair. Two microangiographic Hyodo, and M. Ando, Br. J. Radiol., 1999, 72, 24. sequences were acquired at different rotation angles for 7. K. Umetani, H. Ueki, T. Takeda, Y. Itai, H. Mori, E. Tanaka, stereoscopic observation; and the angle between projections was M. Uddin-Mohammed, Y. Shinozaki, M. Akisada, and Y. approximately 15°. Sasaki, J. Synchrotron Rad., 1998, 5, 1130. Each image is a summation result of five frames. L1 and R1 8. H. Mori, K. Hyodo, E. Tanaka, M. Uddin-Mohammed, A. are summation results from the 16th frame to the 20th frame in Yamakawa, Y. Shinozaki, H. Nakazawa, Y. Tanaka, T. two microangiographic sequences. L2 and R2 are from the 21st- Sekka, Y. Iwata, S. Handa, K. Umetani, H. Ueki, T. 25th, L3 and R3 are from the 26th-30th, and L4 and R4 are from Yokoyama, K. Tanioka, M. Kubota, H. Hosaka, N. Ishikawa, the 31st-35th. Image summation was needed to increase a signal- and M. Ando, Radiology, 1996, 201, 173. to-noise ratio and to visualize small blood vessels. A temporal 9. S. Takeshita, T. Isshiki, H. Mori, E. Tanaka, K. Eto, Y. subtraction operation was also performed for flat field correction Miyazawa, A. Tanaka, Y. Shinozaki, K. Hyodo, M. Ando, using summation results of five frames from the 1st frame to 5th M. Kubota, K. Tanioka, K. Umetani, M. Ochiai, T. Sato, frame, which were taken before the contrast material injection in and H. Miyashita, Circulation, 1997, 95, 805. two microangiographic sequences. Summation images taken 10. K. Umetani, N. Yagi, Y. Suzuki, Y. Ogasawara, F. Kajiya, T. before the injection were subtracted from images corresponding Matsumoto, H. Tachibana, M. Goto, T. Yamashita, S. Imai, to L1-L4 and R1-R4 to eliminate superimposed background and Y. Kajihara, Proc. SPIE, 2000, 3977, 522. structure. 11. K. Umetani, T. Hirai, and S. Takahashi, SPring-8 Annual In Fig. 3, the interval between two consecutive frames is 1.0 s. Report 1999, 2000, 144. Original frames, however, were obtained at a speed of 5 frames/s 12. J. Chikawa, F. Sato, T. Kawamura, T. Kuriyama, T. and an interval of 0.2 s. The 1st and 2nd frames in each sequence Yamashita, and N. Goto, in X-ray Instrumentation for the show the network of tumor-feeding arteries originating from the Photon Factory: Dynamic Analysis of Micro Structures in first-order branch of the proximal auricular artery. The 3rd and Matter, ed. S. Hosoya et al., KTK Scientific Publishers, 4th frames show arteries and veins. In the L1 frame, the Tokyo, 1986, 145. arrowheads show tumor size with a diameter of 7-8 mm. Small 13. H. Kamitsubo, J. Synchrotron Rad., 1998, 5, 162. blood vessels of 20-30 µm diameter were visualized in 14. J. Folkman, J. Natl. Cancer Inst., 1990, 82, 4. consecutive frames. 15. J. Folkman, Nat. Med., 1995, 1, 27. X-ray imaging with limiting spatial resolution of 10-11 µm was carried out using the direct-conversion detector and the nearly parallel X-ray beam provided at the SPring-8. The imaging system could visualize the immature vascular network in the transplanted malignant tumor and was useful for depiction and quantification of angiogenic vessels in the rabbit model of cancer. Stereoscopic images in a monitor screen were observed using a pair of liquid crystal shutter glasses controlled by the digital- image acquisition system. The new system is an effective tool for visualizing three-dimensional structure of tumor-induced