Intravital microscopy of tumor angiogenesis and regression in the dorsal skin fold chamber: mechanistic insights and preclinical testing of therapeutic strategies

  • 626 Accesses

  • 43 Citations


Tumor angiogenesis is a major step in tumor progression to clinically symptomatic cancer and thus a potential target for cancer therapy. It is essential to understand the fundamental mechanisms of the angiogenic processes to provide a rational for testing inhibitory strategies for cancer treatment. The dorsal skin fold chamber provides a suitable (chronic) model for intravital microscopy to monitor the same tumor in time-lapse imaging series and in real-time functional analysis e.g., of blood flow. Adaptation of this model to several rodent species and tumor types has led to numerous physical and drug based therapy options. With modification of implantation techniques, motility and invasion of individual cells can be visualized, in addition to angiogenesis and microcirculation. Modern fluorescent techniques such as ex vivo labelling of specific cell populations and the introduction of stably fluorescent protein expressing cell lines further enhance the suitability of this technique. In addition, laser scanning and multiphoton microscopy in combination with genetically altered mouse strains and cell lines are making the DCSF even more attractive for mechanistic and interventional studies in cancer research. Here we review the preparation as well as the applications of the DCSF in tumor angiogenesis.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 199

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6



Carboxyfluorescein succinimidyl ester


1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate


Dorsal skin fold chamber


Flourescin isothiocyanate


Fluorescence recovery after photobleaching


Functional vascular density (perfused vessels/area)


Green fluorescent protein


Human umbilical cord vein endothelial cells


Human microvascular endothelial cells


Microvasular density (vessel length/area)


Photodynamic therapy


Red blood cell flow


Region of interest


Severe combined immunodeficiency


Tetramethyl rhodamine isothiocyanate


  1. 1.

    Folkman J, Merler E, Abernathy C, Williams G (1971) Isolation of a tumor factor responsible for angiogenesis. J Exp Med 133:275–288. doi:10.1084/jem.133.2.275

  2. 2.

    Kunz-Schughart LA, Schroeder JA, Wondrak M, van Rey F, Lehle K, Hofstaedter F, Wheatley DN (2006) Potential of fibroblasts to regulate the formation of three-dimensional vessel-like structures from endothelial cells in vitro. Am J Physiol Cell Physiol 290:C1385–C1398. doi:10.1152/ajpcell.00248.2005

  3. 3.

    TCS CellWorks BU. 2008

  4. 4.

    Koehl GE, Geissler EK, Iacobelli M, Frei C, Burger V, Haffner S, Holler E, Andreesen R, Schlitt HJ, Eissner G (2007) Defibrotide: an endothelium protecting and stabilizing drug, has an anti-angiogenic potential in vitro and in vivo. Cancer Biol Ther 6:686–690

  5. 5.

    Nicosia RF, McCormick JF, Bielunas J (1984). The formation of endothelial webs and channels in plasma clot culture. Scan Electron Microsc (PT2):793–799

  6. 6.

    Naumov GN, Wilson SM, MacDonald IC, Schmidt EE, Morris VL, Groom AC, Hoffman RM, Chambers AF (1999) Cellular expression of green fluorescent protein, coupled with high-resolution in vivo videomicroscopy, to monitor steps in tumor metastasis. J Cell Sci 112:1835–1842

  7. 7.

    Jain RK, Munn LL, Fukumura D (2002) Dissecting tumour pathophysiology using intravital microscopy. Nat Rev Cancer 2:266–276. doi:10.1038/nrc778

  8. 8.

    Sandison JD (1928) The transparent chamber of the rabbit’s ear giving a complete description of improved techniques of construction and introduction and general account of growth and behavior of living cells and tissues seen with the microscope. Am J Anat 41:447–472. doi:10.1002/aja.1000410303

  9. 9.

    Menger MD, Laschke MW, Vollmar B (2002) Viewing the microcirculation through the window: some twenty years experience with the hamster dorsal skinfold chamber. Eur Surg Res 34:83–91. doi:10.1159/000048893

  10. 10.

    Makale M (2007) Intravital imaging and cell invasion. Methods Enzymol 426:375–401

  11. 11.

    Arfors KE, Jonsson JA, McKenzie FN (1970) A titanium rabbit ear chamber: assembly, insertion and results. Microvasc Res 2:516–518. doi:10.1016/0026-2862(70)90045-2

  12. 12.

    Dellian M, Witwer BP, Salehi HA, Yuan F, Jain RK (1996) Quantitation and physiological characterization of angiogenic vessels in mice: effect of basic fibroblast growth factor, vascular endothelial growth factor/vascular permeability factor, and host microenvironment. Am J Pathol 149:59–71

  13. 13.

    Yuan F, Chen Y, Dellian M, Safabakhsh N, Ferrara N, Jain RK (1996) Time-dependent vascular regression and permeability changes in established human tumor xenografts induced by an anti-vascular endothelial growth factor/vascular permeability factor antibody. Proc Natl Acad Sci USA 93:14765–14770. doi:10.1073/pnas.93.25.14765

  14. 14.

    Hansen-Algenstaedt N, Schaefer C, Wolfram L, Joscheck C, Schroeder M, Algenstaedt P, Ruther W (2005) Femur window—a new approach to microcirculation of living bone in situ. J Orthop Res 23:1073–1082. doi:10.1016/j.orthres.2005.02.013

  15. 15.

    Bertera S, Geng X, Tawadrous Z, Bottino R, Balamurugan AN, Rudert WA, Drain P, Watkins SC, Trucco M (2003) Body window-enabled in vivo multicolor imaging of transplanted mouse islets expressing an insulin-timer fusion protein. BioTechniques 35:718–722

  16. 16.

    Algire GH (1943) An adaptation of the transparent chamber technique to the mouse. J Natl Cancer Inst 4:1–11

  17. 17.

    Zweifach BW (1954) Direct observation of the mesenteric circulation in experimental animals. Anat Rec 120:277–291. doi:10.1002/ar.1091200115

  18. 18.

    Intaglietta M, Tompkins WR, Richardson DR (1970) Velocity measurements in the microvasculature of the cat omentum by on-line method. Microvasc Res 2:462–473. doi:10.1016/0026-2862(70)90039-7

  19. 19.

    Duling BR (1973) The preparation and use of the hamster cheek pouch for studies of the microcirculation. Microvasc Res 5:423–429. doi:10.1016/0026-2862(73)90059-9

  20. 20.

    von Andrian UH (1996) Intravital microscopy of the peripheral lymph node microcirculation in mice. Microcirculation (New York, NY) 3:287–300. doi:10.3109/10739689609148303

  21. 21.

    Fukumura D, Yuan F, Monsky WL, Chen Y, Jain RK (1997) Effect of host microenvironment on the microcirculation of human colon adenocarcinoma. Am J Pathol 151:679–688

  22. 22.

    Wylie S, MacDonald IC, Varghese HJ, Schmidt EE, Morris VL, Groom AC, Chambers AF (1999) The matrix metalloproteinase inhibitor batimastat inhibits angiogenesis in liver metastases of B16F1 melanoma cells. Clin Exp Metastasis 17:111–117. doi:10.1023/A:1006573417179

  23. 23.

    Guba M, Cernaianu G, Koehl G, Geissler EK, Jauch KW, Anthuber M, Falk W, Steinbauer M (2001) A primary tumor promotes dormancy of solitary tumor cells before inhibiting angiogenesis. Cancer Res 61:5575–5579

  24. 24.

    Sahai E, Wyckoff J, Philippar U, Segall JE, Gertler F, Condeelis J (2005) Simultaneous imaging of GFP, CFP and collagen in tumors in vivo using multiphoton microscopy. BMC Biotechnol 5:14

  25. 25.

    Xue C, Wyckoff J, Liang F, Sidani M, Violini S, Tsai KL, Zhang ZY, Sahai E, Condeelis J, Segall JE (2006) Epidermal growth factor receptor overexpression results in increased tumor cell motility in vivo coordinately with enhanced intravasation and metastasis. Cancer Res 66:192–197. doi:10.1158/0008-5472.CAN-05-1242

  26. 26.

    Nolte D, Menger MD, Messmer K (1995) Microcirculatory models of ischaemia-reperfusion in skin and striated muscle. Int J Microcirc Clin Exp 15(Suppl 1):9–16

  27. 27.

    Harder Y, Amon M, Erni D, Menger MD (2004) Evolution of ischemic tissue injury in a random pattern flap: a new mouse model using intravital microscopy. J Surg Res 121:197–205. doi:10.1016/j.jss.2004.03.026

  28. 28.

    Funk W, Endrich B, Messmer K (1986) A novel method for follow-up studies of the microcirculation in non-malignant tissue implants. Res Exp Med (Berl) 186:259–270. doi:10.1007/BF01852303

  29. 29.

    Leunig M, Yuan F, Berk DA, Gerweck LE, Jain RK (1994) Angiogenesis and growth of isografted bone: quantitative in vivo assay in nude mice. Lab Invest 71:300–307

  30. 30.

    Vajkoczy P, Menger MD, Simpson E, Messmer K (1995) Angiogenesis and vascularization of murine pancreatic islet isografts. Transplantation 60:123–127

  31. 31.

    Vollmar B, Laschke MW, Rohan R, Koenig J, Menger MD (2001) In vivo imaging of physiological angiogenesis from immature to preovulatory ovarian follicles. Am J Pathol 159:1661–1670

  32. 32.

    Menger MD, Hammersen F, Walter P, Messmer K (1990) Neovascularization of prosthetic vascular grafts. Quantitative analysis of angiogenesis and microhemodynamics by means of intravital microscopy. Thorac Cardiovasc Surg 38:139–145. doi:10.1055/s-2007-1014008

  33. 33.

    Rucker M, Laschke MW, Junker D, Carvalho C, Tavassol F, Mulhaupt R, Gellrich NC, Menger MD (2007) Vascularization and biocompatibility of scaffolds consisting of different calcium phosphate compounds. J Biomed Mater Res A 86:1002–1011

  34. 34.

    Schumann D (2004) Methoden zur Optimierung von tissue engineering Produkten auf dem Wege zur Reparatur osteochondraler Defekte. Dissertation, Naturwissenschaftliche Fakultät IV—Chemie und Pharmazie, University of Regensburg

  35. 35.

    Asaishi K, Endrich B, Gotz A, Messmer K (1981) Quantitative analysis of microvascular structure and function in the amelanotic melanoma A-Mel-3. Cancer Res 41:1898–1904

  36. 36.

    Papenfuss HD, Gross JF, Intaglietta M, Treese FA (1979) A transparent access chamber for the rat dorsal skin fold. Microvasc Res 18:311–318. doi:10.1016/0026-2862(79)90039-6

  37. 37.

    Wu NZ, Ross BA, Gulledge C, Klitzman B, Dodge R, Dewhirst MW (1994) Differences in leucocyte-endothelium interactions between normal and adenocarcinoma bearing tissues in response to radiation. Br J Cancer 69:883–889

  38. 38.

    Bruns CJ, Koehl GE, Guba M, Yezhelyev M, Steinbauer M, Seeliger H, Schwend A, Hoehn A, Jauch KW, Geissler EK (2004) Rapamycin-induced endothelial cell death and tumor vessel thrombosis potentiate cytotoxic therapy against pancreatic cancer. Clin Cancer Res 10:2109–2119. doi:10.1158/1078-0432.CCR-03-0502

  39. 39.

    Szczesny G, Veihelmann A, Massberg S, Nolte D, Messmer K (2004) Long-term anaesthesia using inhalatory isoflurane in different strains of mice-the haemodynamic effects. Lab Anim 38:64–69. doi:10.1258/00236770460734416

  40. 40.

    Berk DA, Yuan F, Leunig M, Jain RK (1997) Direct in vivo measurement of targeted binding in a human tumor xenograft. Proc Natl Acad Sci USA 94:1785–1790. doi:10.1073/pnas.94.5.1785

  41. 41.

    Pluen A, Boucher Y, Ramanujan S, McKee TD, Gohongi T, di Tomaso E, Brown EB, Izumi Y, Campbell RB, Campbell RB, Berk DA, Jain RK (2001) Role of tumor-host interactions in interstitial diffusion of macromolecules: cranial vs. subcutaneous tumors. Proc Natl Acad Sci USA 98:4628–4633. doi:10.1073/pnas.081626898

  42. 42.

    Strieth S, Nussbaum CF, Eichhorn ME, Fuhrmann M, Teifel M, Michaelis U, Berghaus A, Dellian M (2008) Tumor-selective vessel occlusions by platelets after vascular targeting chemotherapy using paclitaxel encapsulated in cationic liposomes. Int J Cancer 122:452–460. doi:10.1002/ijc.23088

  43. 43.

    Dreher MR, Liu W, Michelich CR, Dewhirst MW, Yuan F, Chilkoti A (2006) Tumor vascular permeability, accumulation, and penetration of macromolecular drug carriers. J Natl Cancer Inst 98:335–344

  44. 44.

    Reyes-Aldasoro CC, Wilson I, Prise VE, Barber PR, Ameer-Beg M, Vojnovic B, Cunningham VJ, Tozer GM (2008) Estimation of apparent tumor vascular permeability from multiphoton fluorescence microscopic images of P22 rat sarcomas in vivo. Microcirculation (New York, NY) 15:65–79. doi:10.1080/10739680701436350

  45. 45.

    Alexander S, Koehl GE, Hierschberg M, Geissler EK, Friedl P (2008) Dynamic imaging of cancer growth and invasion: a modified skin-fold chamber model. Histochem Cell Biol 130:1147–1154

  46. 46.

    Endrich B, Asaishi K, Gotz A, Messmer K (1980) Technical report—a new chamber technique for microvascular studies in unanesthetized hamsters. Res Exp Med (Berl) 177:125–134. doi:10.1007/BF01851841

  47. 47.

    Schacht V, Berens von Rautenfeld D, Abels C (2004) The lymphatic system in the dorsal skinfold chamber of the Syrian golden hamster in vivo. Arch Dermatol Res 295:542–548. doi:10.1007/s00403-004-0453-8

  48. 48.

    Isaka N, Padera TP, Hagendoorn J, Fukumura D, Jain RK (2004) Peritumor lymphatics induced by vascular endothelial growth factor-C exhibit abnormal function. Cancer Res 64:4400–4404. doi:10.1158/0008-5472.CAN-04-0752

  49. 49.

    Leunig M, Yuan F, Menger MD, Boucher Y, Goetz AE, Messmer K, Jain RK (1992) Angiogenesis, microvascular architecture, microhemodynamics, and interstitial fluid pressure during early growth of human adenocarcinoma LS174T in SCID mice. Cancer Res 52:6553–6560

  50. 50.

    Lehr HA, Leunig M, Menger MD, Nolte D, Messmer K (1993) Dorsal skinfold chamber technique for intravital microscopy in nude mice. Am J Pathol 143:1055–1062

  51. 51.

    Steinbauer M, Harris AG, Abels C, Messmer K (2000) Characterization and prevention of phototoxic effects in intravital fluorescence microscopy in the hamster dorsal skinfold model. Langenbecks Arch Surg 385:290–298. doi:10.1007/s004239900108

  52. 52.

    Patumraj S, Yoysungnoen P, Kachonrattanadet P, Wirachwong P (2005) Tumor neocapillary density in hepatocellular carcinoma cells implanted nude mice model. Clin Hemorheol Microcirc 33:137–144

  53. 53.

    Wu NZ, Klitzman B, Rosner G, Needham D, Dewhirst MW (1993) Measurement of material extravasation in microvascular networks using fluorescence video-microscopy. Microvasc Res 46:231–253. doi:10.1006/mvre.1993.1049

  54. 54.

    Nugent LJ, Jain RK (1984) Plasma pharmacokinetics and interstitial diffusion of macromolecules in a capillary bed. Am J Physiol 246:H129–H137

  55. 55.

    Yuan F, Leunig M, Berk DA, Jain RK (1993) Microvascular permeability of albumin, vascular surface area, and vascular volume measured in human adenocarcinoma LS174T using dorsal chamber in SCID mice. Microvasc Res 45:269–289. doi:10.1006/mvre.1993.1024

  56. 56.

    Yuan F, Dellian M, Fukumura D, Leunig M, Berk DA, Torchilin VP, Jain RK (1995) Vascular permeability in a human tumor xenograft: molecular size dependence and cutoff size. Cancer Res 55:3752–3756

  57. 57.

    Hobbs SK, Monsky WL, Yuan F, Roberts WG, Griffith L, Torchilin VP, Jain RK (1998) Regulation of transport pathways in tumor vessels: role of tumor type and microenvironment. Proc Natl Acad Sci USA 95:4607–4612. doi:10.1073/pnas.95.8.4607

  58. 58.

    Yuan F, Leunig M, Huang SK, Berk DA, Papahadjopoulos D, Jain RK (1994) Microvascular permeability and interstitial penetration of sterically stabilized (stealth) liposomes in a human tumor xenograft. Cancer Res 54:3352–3356

  59. 59.

    Gerlowski LE, Jain RK (1986) Microvascular permeability of normal and neoplastic tissues. Microvasc Res 31:288–305. doi:10.1016/0026-2862(86)90018-X

  60. 60.

    Kohn S, Nagy JA, Dvorak HF, Dvorak AM (1992) Pathways of macromolecular tracer transport across venules and small veins. Structural basis for the hyperpermeability of tumor blood vessels. Lab Invest 67:596–607

  61. 61.

    Fukumura D, Yuan F, Endo M, Jain RK (1997) Role of nitric oxide in tumor microcirculation. Blood flow, vascular permeability, and leukocyte–endothelial interactions. Am J Pathol 150:713–725

  62. 62.

    Koehl G, Guba M, Seeliger H, Steinbauer M, Anthuber M, Jauch KW, Geissler EK (2003) Rapamycin treatment at immunosuppressive doses affects tumor blood vessel circulation. Transplant Proc 35:2135–2136. doi:10.1016/S0041-1345(03)00745-0

  63. 63.

    Saetzler RK, Jallo J, Lehr HA, Philips CM, Vasthare U, Arfors KE, Tuma RF (1997) Intravital fluorescence microscopy: impact of light-induced phototoxicity on adhesion of fluorescently labeled leukocytes. J Histochem Cytochem 45:505–513

  64. 64.

    Baatz H, Steinbauer M, Harris AG, Krombach F (1995) Kinetics of white blood cell staining by intravascular administration of rhodamine 6G. Int J Microcirc Clin Exp 15:85–91

  65. 65.

    Harris AG, Steinbauer M, Leiderer R, Messmer K (1997) Role of leukocyte plugging and edema in skeletal muscle ischemia-reperfusion injury. Am J Physiol 273:H989–H996

  66. 66.

    Vajkoczy P, Goldbrunner R, Farhadi M, Vince G, Schilling L, Tonn JC, Schmiedek P, Menger MD (1999) Glioma cell migration is associated with glioma-induced angiogenesis in vivo. Int J Dev Neurosci 17:557–563. doi:10.1016/S0736-5748(99)00021-0

  67. 67.

    Guba M, Yezhelyev M, Eichhorn ME, Schmid G, Ischenko I, Papyan A, Graeb C, Seeliger H, Geissler EK, Jauch KW, Bruns CJ (2005) Rapamycin induces tumor-specific thrombosis via tissue factor in the presence of VEGF. Blood 105:4463–4469. doi:10.1182/blood-2004-09-3540

  68. 68.

    Lang SA, Gaumann A, Koehl GE, Seidel U, Bataille F, Klein D, Ellis LM, Bolder U, Hofstaedter F, Schlitt HJ, Geissler EK, Stoeltzing O (2007) Mammalian target of rapamycin is activated in human gastric cancer and serves as a target for therapy in an experimental model. Int J Cancer 120:1803–1810

  69. 69.

    Mordon S, Begu S, Buys B, Tourne-Peteilh C, Devoisselle JM (2002) Study of platelet behavior in vivo after endothelial stimulation with laser irradiation using fluorescence intravital videomicroscopy and PEGylated liposome staining. Microvasc Res 64:316–325. doi:10.1006/mvre.2002.2435

  70. 70.

    Cernaianu G, Frank S, Erbstosser K, Leonhardt S, Cross M, McIvor Z, Scholz G, Dansranjavin T, Celik I, Tannapfel A, Wittekind C, Troebs RB, Rothe K, Bennek J, Hauss J, Witzigmann H (2006) TNP-470 fails to block the onset of angiogenesis and early tumor establishment in an intravital minimal disease model. Int J Colorectal Dis 21:143–154. doi:10.1007/s00384-005-0751-4

  71. 71.

    Yamauchi K, Yang M, Jiang P, Xu M, Yamamoto N, Tsuchiya H, Tomita K, Moossa AR, Bouvet M, Hoffman RM (2006) Development of real-time subcellular dynamic multicolor imaging of cancer-cell trafficking in live mice with a variable-magnification whole-mouse imaging system. Cancer Res 66:4208–4214. doi:10.1158/0008-5472.CAN-05-3927

  72. 72.

    Cao Y, Li CY, Moeller BJ, Yu D, Zhao Y, Dreher MR, Shan S, Dewhirst MW (2005) Observation of incipient tumor angiogenesis that is independent of hypoxia and hypoxia inducible factor-1 activation. Cancer Res 65:5498–5505. doi:10.1158/0008-5472.CAN-04-4553

  73. 73.

    Turner J, Rhee JG, Fabian DF, Lefor AT (1997) Expression of ICAM-1 enhances in vivo lymphocyte adhesion in a murine fibrosarcoma. J Surg Oncol 66:39–44. doi:10.1002/(SICI)1096-9098(199709)66:1<39::AID-JSO8>3.0.CO;2-O

  74. 74.

    Babilas P, Shafirstein G, Baumler W, Baier J, Landthaler M, Szeimies RM, Abels C (2005) Selective photothermolysis of blood vessels following flashlamp-pumped pulsed dye laser irradiation: in vivo results and mathematical modelling are in agreement. J Invest Dermatol 125:343–352

  75. 75.

    Babilas P, Shafirstein G, Baier J, Schacht V, Szeimies RM, Landthaler M, Baumler W, Abels C (2007) Photothermolysis of blood vessels using indocyanine green and pulsed diode laser irradiation in the dorsal skinfold chamber model. Lasers Surg Med 39:341–352. doi:10.1002/lsm.20483

  76. 76.

    Schacht V, Becker K, Szeimies RM, Abels C (2002) Apoptosis and leucocyte-endothelium interactions contribute to the delayed effects of cryotherapy on tumours in vivo. Arch Dermatol Res 294:341–348

  77. 77.

    Hoffmann NE, Bischof JC (2001) Cryosurgery of normal and tumor tissue in the dorsal skin flap chamber: part II–injury response. J Biomech Eng 123:310–316. doi:10.1115/1.1385839

  78. 78.

    Bhowmick S, Hoffmann NE, Bischof JC (2002) Thermal therapy of prostate tumor tissue in the dorsal skin flap chamber. Microvasc Res 64:170–173. doi:10.1006/mvre.2002.2408

  79. 79.

    Abels C, Heil P, Dellian M, Kuhnle GE, Baumgartner R, Goetz AE (1994) In vivo kinetics and spectra of 5-aminolaevulinic acid-induced fluorescence in an amelanotic melanoma of the hamster. Br J Cancer 70:826–833

  80. 80.

    Dolmans DE, Kadambi A, Hill JS, Waters CA, Robinson BC, Walker JP, Fukumura D, Jain RK (2002) Vascular accumulation of a novel photosensitizer, MV6401, causes selective thrombosis in tumor vessels after photodynamic therapy. Cancer Res 62:2151–2156

  81. 81.

    Kruijt B, de Bruijn HS, van der Ploeg-van den Heuvel A, Sterenborg HJ, Robinson DJ (2006) Laser speckle imaging of dynamic changes in flow during photodynamic therapy. Lasers Med Sci 21:208–212. doi:10.1007/s10103-006-0399-5

  82. 82.

    Hori K, Saito S, Tamai M (2004) Effect of irradiation on neovascularization in rat skinfold chambers: implications for clinical trials of low-dose radiotherapy for wet-type age-related macular degeneration. Int J Radiat Oncol Biol Phys 60:1564–1571. doi:10.1016/j.ijrobp.2004.06.208

  83. 83.

    Endrich B, Goetz A, Messmer K (1982) Distribution of microflow and oxygen tension in hamster melanoma. Int J Microcirc Clin Exp 1:81–99

  84. 84.

    Dewhirst MW, Ong ET, Braun RD, Smith B, Klitzman B, Evans SM, Wilson D (1999) Quantification of longitudinal tissue pO2 gradients in window chamber tumours: impact on tumour hypoxia. Br J Cancer 79:1717–1722. doi:10.1038/sj.bjc.6690273

  85. 85.

    Makale MT, Lin JT, Calou RE, Tsai AG, Chen PC, Gough DA (2003) Tissue window chamber system for validation of implanted oxygen sensors. Am J Physiol Heart Circ Physiol 284:H2288–H2294

  86. 86.

    Babilas P, Liebsch G, Schacht V, Klimant I, Wolfbeis OS, Szeimies RM, Abels C (2005) In vivo phosphorescence imaging of pO2 using planar oxygen sensors. Microcirculation (New York, NY) 12:477–487. doi:10.1080/10739680591003314

  87. 87.

    Dewhirst MW, Ong ET, Klitzman B, Secomb TW, Vinuya RZ, Dodge R, Brizel D, Gross JF (1992) Perivascular oxygen tensions in a transplantable mammary tumor growing in a dorsal flap window chamber. Radiat Res 130:171–182. doi:10.2307/3578274

  88. 88.

    Kerger H, Torres FI, Rivas M, Winslow RM, Intaglietta M (1995) Systemic and subcutaneous microvascular oxygen tension in conscious Syrian golden hamsters. Am J Physiol 268:H802–H810

  89. 89.

    Monsky WL, Fukumura D, Gohongi T, Ancukiewcz M, Weich HA, Torchilin VP, Yuan F, Jain RK (1999) Augmentation of transvascular transport of macromolecules and nanoparticles in tumors using vascular endothelial growth factor. Cancer Res 59:4129–4135

  90. 90.

    Griffon-Etienne G, Boucher Y, Brekken C, Suit HD, Jain RK (1999) Taxane-induced apoptosis decompresses blood vessels and lowers interstitial fluid pressure in solid tumors: clinical implications. Cancer Res 59:3776–3782

  91. 91.

    Hansen-Algenstaedt N, Stoll BR, Padera TP, Dolmans DE, Hicklin DJ, Fukumura D, Jain RK (2000) Tumor oxygenation in hormone-dependent tumors during vascular endothelial growth factor receptor-2 blockade, hormone ablation, and chemotherapy. Cancer Res 60:4556–4560

  92. 92.

    Sehgal SN (2003) Sirolimus: its discovery, biological properties, and mechanism of action. Transplant Proc 35:7S–14S. doi:10.1016/S0041-1345(03)00211-2

  93. 93.

    Sehgal SN (1998) Rapamune (RAPA, rapamycin, sirolimus): mechanism of action immunosuppressive effect results from blockade of signal transduction and inhibition of cell cycle progression. Clin Biochem 31:335–340. doi:10.1016/S0009-9120(98)00045-9

  94. 94.

    Augustine JJ, Bodziak KA, Hricik DE (2007) Use of sirolimus in solid organ transplantation. Drugs 67:369–391. doi:10.2165/00003495-200767030-00004

  95. 95.

    Koehl GE, Schlitt HJ, Geissler EK (2005) Rapamycin and tumor growth: mechanisms behind its anticancer activity. Transplant Rev 19:20–31. doi:10.1016/j.trre.2005.01.001

  96. 96.

    Gaumann A, Schlitt HJ, Geissler EK (2008) Immunosuppression and tumor development in organ transplant recipients: the emerging dualistic role of rapamycin. Transpl Int 21:207–217. doi:10.1111/j.1432-2277.2007.00610.x

  97. 97.

    Geissler EK, Schlitt HJ (2004) The relation between immunosuppressive agents and malignancy. Curr Opin Organ Transplant 9:394–399. doi:10.1097/01.mot.0000146559.20280.29

  98. 98.

    Guba M, von Breitenbuch P, Steinbauer M, Koehl G, Flegel S, Hornung M, Bruns CJ, Zuelke C, Farkas S, Anthuber M, Jauch KW, Geissler EK (2002) Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor. Nat Med 8:128–135. doi:10.1038/nm0202-128

  99. 99.

    Baish JW, Gazit Y, Berk DA, Nozue M, Baxter LT, Jain RK (1996) Role of tumor vascular architecture in nutrient and drug delivery: an invasion percolation-based network model. Microvasc Res 51:327–346. doi:10.1006/mvre.1996.0031

  100. 100.

    Lichtenbeld HC, Barendsz-Janson AF, van Essen H, Struijker BH, Griffioen AW, Hillen HF (1998) Angiogenic potential of malignant and non-malignant human breast tissues in an in vivo angiogenesis model. Int J Cancer 77:455–459. doi:10.1002/(SICI)1097-0215(19980729)77:3<455::AID-IJC23>3.0.CO;2-5

  101. 101.

    Koehl GE, Wagner F, Stoeltzing O, Lang SA, Steinbauer M, Schlitt HJ, Geissler EK (2007) Mycophenolate mofetil inhibits tumor growth and angiogenesis in vitro but has variable antitumor effects in vivo, possibly related to bioavailability. Transplantation 83:607–614. doi:10.1097/

  102. 102.

    Laschke MW, Elitzsch A, Scheuer C, Vollmar B, Menger MD (2007) Selective cyclo-oxygenase-2 inhibition induces regression of autologous endometrial grafts by down-regulation of vascular endothelial growth factor-mediated angiogenesis and stimulation of caspase-3-dependent apoptosis. Fertil Steril 87:163–171. doi:10.1016/j.fertnstert.2006.05.068

  103. 103.

    Laschke MW, Elitzsch A, Scheuer C, Holstein JH, Vollmar B, Menger MD (2006) Rapamycin induces regression of endometriotic lesions by inhibiting neovascularization and cell proliferation. Br J Pharmacol 149:137–144. doi:10.1038/sj.bjp.0706857

  104. 104.

    Laschke MW, Elitzsch A, Vollmar B, Vajkoczy P, Menger MD (2006) Combined inhibition of vascular endothelial growth factor (VEGF), fibroblast growth factor and platelet-derived growth factor, but not inhibition of VEGF alone, effectively suppresses angiogenesis and vessel maturation in endometriotic lesions. Hum Reprod 21:262–268. doi:10.1093/humrep/dei308

  105. 105.

    Manegold PC, Hutter J, Pahernik SA, Messmer K, Dellian M (2003) Platelet–endothelial interaction in tumor angiogenesis and microcirculation. Blood 101:1970–1976. doi:10.1182/blood.V101.5.1970

  106. 106.

    Griffin RJ, Williams BW, Bischof JC, Olin M, Johnson GL, Lee BW (2007) Use of a fluorescently labeled poly-caspase inhibitor for in vivo detection of apoptosis related to vascular-targeting agent arsenic trioxide for cancer therapy. Technol Cancer Res Treat 6:651–654

  107. 107.

    Vajkoczy P, Menger MD, Goldbrunner R, Ge S, Fong TA, Vollmar B, Schilling L, Ullrich A, Hirth KP, Tonn JC, Schmiedek P, Rempel SA (2000) Targeting angiogenesis inhibits tumor infiltration and expression of the pro-invasive protein SPARC. Int J Cancer 87:261–268. doi:10.1002/1097-0215(20000715)87:2<261::AID-IJC18>3.0.CO;2-6

  108. 108.

    Vajkoczy P, Thurnher A, Hirth KP, Schilling L, Schmiedek P, Ullrich A, Menger MD (2000) Measuring VEGF-Flk-1 activity and consequences of VEGF-Flk-1 targeting in vivo using intravital microscopy: clinical applications. Oncologist 5(Suppl 1):16–19

  109. 109

    Laird AD, Christensen JG, Li G, Carver J, Smith K, Xin X, Moss KG, Louie SG, Mendel DB, Cherrington JM (2002) SU6668 inhibits Flk–1/KDR and PDGFRbeta in vivo, resulting in rapid apoptosis of tumor vasculature and tumor regression in mice. FASEB J 16:681–690

  110. 110.

    Yoysungnoen P, Wirachwong P, Bhattarakosol P, Niimi H, Patumraj S (2005) Antiangiogenic activity of curcumin in hepatocellular carcinoma cells implanted nude mice. Clin Hemorheol Microcirc 33:127–135

  111. 111.

    Yoysungnoen P, Wirachwong P, Bhattarakosol P, Niimi H, Patumraj S (2006) Effects of curcumin on tumor angiogenesis and biomarkers, COX-2 and VEGF, in hepatocellular carcinoma cell-implanted nude mice. Clin Hemorheol Microcirc 34:109–115

  112. 112.

    Borgstrom P, Torres FI, Hartley-Asp B (1995) Inhibition of angiogenesis and metastases of the Lewis-lung cell carcinoma by the quinoline-3-carboxamide. Linomide Anticancer Res 15:719–728

  113. 113.

    Torres FI, Hartley-Asp B, Borgstrom P (1995) Quantitative angiogenesis in a syngeneic tumor spheroid model. Microvasc Res 49:212–226. doi:10.1006/mvre.1995.1017

  114. 114.

    Levin EG, Sikora L, Ding L, Rao SP, Sriramarao P (2004) Suppression of tumor growth and angiogenesis in vivo by a truncated form of 24-kd fibroblast growth factor (FGF)-2. Am J Pathol 164:1183–1190

  115. 115.

    de Bruijn HS, Kruijt B, van der Ploeg-van den Heuvel A, Sterenborg HJ, Robinson DJ (2007) Increase in protoporphyrin IX after 5-aminolevulinic acid based photodynamic therapy is due to local re-synthesis. Photochem Photobiol Sci 6:857–864. doi:10.1039/b703361c

  116. 116.

    Shan S, Lockhart AC, Saito WY, Knapp AM, Laderoute KR, Dewhirst MW (2001) The novel tubulin-binding drug BTO-956 inhibits R3230AC mammary carcinoma growth and angiogenesis in Fischer 344 rats. Clin Cancer Res 7:2590–2596

  117. 117.

    Erickson K, Braun RD, Yu D, Lanzen J, Wilson D, Brizel DM, Secomb TW, Biaglow JE, Dewhirst MW (2003) Effect of longitudinal oxygen gradients on effectiveness of manipulation of tumor oxygenation. Cancer Res 63:4705–4712

  118. 118.

    Ackermann G, Abels C, Baumler W, Langer S, Landthaler M, Lang EW, Szeimies RM (1998) Simulations on the selectivity of 5-aminolaevulinic acid-induced fluorescence in vivo. J Photochem Photobiol B 47:121–128. doi:10.1016/S1011-1344(98)00210-3

  119. 119.

    Schacht V, Szeimies RM, Abels C (2006) Photodynamic therapy with 5-aminolevulinic acid induces distinct microcirculatory effects following systemic or topical application. Photochem Photobiol Sci 5:452–458. doi:10.1039/b514128a

  120. 120.

    Eichhorn ME, Luedemann S, Strieth S, Papyan A, Ruhstorfer H, Haas H, Michaelis U, Sauer B, Teifel M, Enders G, Brix G, Jauch KW, Bruns CJ, Dellian M (2007) Cationic lipid complexed camptothecin (EndoTAG-2) improves antitumoral efficacy by tumor vascular targeting. Cancer Biol Ther 6:920–929

  121. 121.

    Endrich B, Hammersen F, Gotz A, Messmer K (1982) Microcirculatory blood flow, capillary morphology and local oxygen pressure of the hamster amelanotic melanoma A-Mel-3. J Natl Cancer Inst 68:475–485

  122. 122.

    Babilas P, Schacht V, Liebsch G, Wolfbeis OS, Landthaler M, Szeimies RM, Abels C (2003) Effects of light fractionation and different fluence rates on photodynamic therapy with 5-aminolaevulinic acid in vivo. Br J Cancer 88:1462–1469. doi:10.1038/sj.bjc.6600910

  123. 123.

    Kleespies A, Kohl G, Friedrich M, Ryan AJ, Barge A, Jauch KW, Bruns CJ (2005) Vascular targeting in pancreatic cancer: the novel tubulin-binding agent ZD6126 reveals antitumor activity in primary and metastatic tumor models. Neoplasia (New York, NY) 7:957–966. doi:10.1593/neo.05304

  124. 124.

    Conrad C, Ischenko I, Kohl G, Wiegand U, Guba M, Yezhelyev M, Ryan AJ, Barge A, Geissler EK, Wedge SR, Jauch KW, Bruns CJ (2007) Antiangiogenic and antitumor activity of a novel vascular endothelial growth factor receptor-2 tyrosine kinase inhibitor ZD6474 in a metastatic human pancreatic tumor model. Anticancer Drugs 18:569–579. doi:10.1097/CAD.0b013e3280147d13

  125. 125.

    Sunamura M, Sun L, Lozonschi L, Duda DG, Kodama T, Matsumoto G, Shimamura H, Takeda K, Kobari M, Hamada H, Matsuno S (2000) The antiangiogenesis effect of interleukin 12 during early growth of human pancreatic cancer in SCID mice. Pancreas 20:227–233. doi:10.1097/00006676-200004000-00002

  126. 126.

    Heuser M, Schlott T, Schally AV, Kahler E, Schliephaker R, Laabs SO, Hemmerlein B (2005) Expression of gastrin releasing Peptide receptor in renal cell carcinomas: a potential function for the regulation of neoangiogenesis and microvascular perfusion. J Urol 173:2154–2159. doi:10.1097/01.ju.0000158135.26893.bc

Download references


We would like to thank several people for their contributions. First, we want to thank our many past and present colleagues at the University of Regensburg for their contributions, including especially Dr. Markus Steinbauer and Dr. Markus Guba for their expertise with the DSFC model. We also appreciate collaborations with Dr. Oliver Stoeltzing, Dr. Sven Lang and Dr. Christiane Bruns, who allowed adaptation of their tumor models to the DSFC system. Furthermore, we appreciate the efforts of Dr. Ferdinand Wagner in helping to provide images for this publication. Finally, we want to thank Dr. Philipp Babilas (Department of Dermatology) for Fig. 4, and for valuable comments.

Author information

Correspondence to Gudrun E. Koehl.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 2 (MPG 4051 kb)

Supplementary material 3 (MPG 2946 kb)

Supplementary material 4 (MPG 4868 kb)

Supplementary material 5 (MPG 5202 kb)

Supplementary material 6 (MPG 4818 kb)

Supplementary material 7 (MPG 4818 kb)

Supplementary material 8 (MPG 4722 kb)

Supplementary material 1 (DOC 23 kb)

Supplementary material 2 (MPG 4051 kb)

Supplementary material 3 (MPG 2946 kb)

Supplementary material 4 (MPG 4868 kb)

Supplementary material 5 (MPG 5202 kb)

Supplementary material 6 (MPG 4818 kb)

Supplementary material 7 (MPG 4818 kb)

Supplementary material 8 (MPG 4722 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Koehl, G.E., Gaumann, A. & Geissler, E.K. Intravital microscopy of tumor angiogenesis and regression in the dorsal skin fold chamber: mechanistic insights and preclinical testing of therapeutic strategies. Clin Exp Metastasis 26, 329–344 (2009).

Download citation


  • Cancer
  • Tumor angiogenesis
  • Blood vessel
  • Blood flow
  • Microcirculation
  • Dorsal skin fold chamber
  • Fluorescence microscopy
  • Intravital microscopy
  • Tumor therapy