An improved procedure to quantify tumour vascularity using true colour image analysis. Comparison with the manual hot-spot procedure in a human melanoma xenograft model

Laak, J.A.W.M. van der; Westphal, Johan R.; Schalkwijk, Lia; Pahlplatz, M. M. M.; Ruiter, Dirk J.; Waal, R.M.W. de; Wilde, P.C.M. de

doi:10.1002/(sici)1096-9896(199802)184:2<136::aid-path970>3.0.co;2-9

Cited by 43 publications

(32 citation statements)

References 16 publications

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“…Consistent with this assumption, immunohistochemical analysis of tissue sections of CDT6 expressing xenografts stained for blood vessel endothelial cells indeed showed aberrant blood vessels. The blood vessel diameter and lumen was significantly increased as determined by automated image analysis (12). From these results we initially assumed that similar to angiopoietin-1 and angiopoietin-2, CDT6 was involved in vascular morphogenesis.…”

Section: Discussionmentioning

confidence: 99%

“…Fixation and immunocytochemical analysis was performed on cultured BLM cells in the same manner as that described for cryosections of xenografts. The VIDAS image analysis system was used to determine number of vessels per surface unit and blood vessel area and diameter (12). Proteoglycans were visualized by conventional Alcian blue or Azan staining.…”

Section: Methodsmentioning

confidence: 99%

“…Ten g of RNA was size-fractionated on a 1% agarose, 2 M formaldehyde gel in MOPS buffer and blotted to Hybond N filter in 10ϫ SSC after transfer blots were hybridized with random prime-labeled cDNA probes. For reverse transcriptase-PCR analysis, 5 g of total RNA was reverse-transcribed in the presence of oligo(dT) [12][13][14][15][16][17][18] and 200 units of Superscript Range H reverse transcriptase (Invitrogen) according to the manufacturers' instructions. PCR was performed in a thermocycler (PerkinElmer Life Sciences) for 23 cycles.…”

Section: Methodsmentioning

confidence: 99%

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The Angiopoietin-like Factor Cornea-derived Transcript 6 Is a Putative Morphogen for Human Cornea

Peek¹,

Kammerer²,

Frank³

et al. 2002

Journal of Biological Chemistry

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The human cornea-specific protein cornea-derived transcript 6 (CDT6) is a member of the angiopoietin gene family. We report the structural and functional characterization of CDT6. We demonstrate that CDT6 is a secreted protein that folds into disulfide-linked homotetramers by coiled-coil interactions. The finding that CDT6 is expressed at high levels in the avascular corneal stromal layer suggested that the protein, similar to certain angiopoietins, acts as a negative regulator of angiogenesis. To test this hypothesis and to assay the effect of the protein on a growing tissue with high vascular density, CDT6 was expressed in a mouse xenograft model. Expression of CDT6 led to a reduction in tumor growth and aberrant blood vessel formation by inducing massive fibrosis. Interestingly, expression of CDT6 also resulted in the deposition of extracellular matrix components typical for the mature corneal stromal layer. These observations strongly suggest a role as morphogen for CDT6 in inducing a corneal phenotype in vivo.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The Angiopoietin-like Factor Cornea-derived Transcript 6 Is a Putative Morphogen for Human Cornea

Peek¹,

Kammerer²,

Frank³

et al. 2002

Journal of Biological Chemistry

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“…The extent of blood vessel development in a tumor was evaluated based on the total length of blood vessels (L) in chosen areas: areas containing the highest number of vessels were identified by scanning the tumors (28) by intravital or whole-body imaging. To compare the level of vascularization during tumor growth, the ''hot'' areas with the maximum development of vessels per unit area (35) were then quantitated for L expressed in pixels. Captured images were corrected for unevenness in illumination.…”

Section: Soi Of Tumors (23)mentioning

confidence: 99%

Whole-body and intravital optical imaging of angiogenesis in orthotopically implanted tumors

Yang

Baranov

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

131

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The development of drugs for the control of tumor angiogenesis requires a simple, accurate, and economical assay for tumorinduced vascularization. We have adapted the orthotopic implantation model to angiogenesis measurement by using human tumors labeled with Aequorea victoria green fluorescent protein for grafting into nude mice. The nonluminous induced capillaries are clearly visible against the very bright tumor fluorescence examined either intravitally or by whole-body luminance in real time. The orthotopic implantation model of human cancer has been well characterized, and fluorescence shadowing replaces the laborious histological techniques for determining blood vessel density. Intravital images of orthotopically implanted human pancreatic tumors clearly show angiogenic capillaries at both primary and metastatic sites. A quantitative time course of angiogenesis was determined for an orthotopically growing human prostate tumor periodically imaged intravitally in a single nude mouse over a 19-day period. Whole-body optical imaging of tumor angiogenesis was demonstrated by injecting fluorescent Lewis lung carcinoma cells into the s.c. site of the footpad of nude mice. The footpad is relatively transparent, with comparatively few resident blood vessels, allowing quantitative imaging of tumor angiogenesis in the intact animal. Capillary density increased linearly over a 10-day period as determined by whole-body imaging. Similarly, the green fluorescent protein-expressing human breast tumor MDA-MB-435 was orthotopically transplanted to the mouse fat pad, where whole-body optical imaging showed that blood vessel density increased linearly over a 20-week period. These powerful and clinically relevant angiogenesis mouse models can be used for real-time in vivo evaluation of agents inhibiting or promoting tumor angiogenesis in physiological microenvironments.green fluorescent protein ͉ metastatic models ͉ external optical imaging T umor vascularization or angiogenesis is a critical step in tumor growth, progression, and metastasis. As such, angiogenesis promises a uniquely effective yet remarkably benign target for cancer chemotherapy. A major requirement for the effective discovery of angiogenesis-related drugs is an assay system that is accurate, rapid, and economical. We report a model system that meets these requirements.The discovery and evaluation of antiangiogenic substances initially relied on in vivo methods such as the chorioallantoic membrane assay (1, 2), the monkey iris neovascularization model (3), the disk angiogenesis assay (4), and various models that use the cornea to assess blood vessel growth (5-10). Although they are important for understanding the mechanisms of blood vessel induction, these models did not deal with tumor angiogenesis and are poorly suited to drug discovery.Subcutaneous tumor xenograft angiogenesis models have been developed to study tumor angiogenesis, but these require cumbersome pathological examination procedures such as histology and immunohistochemistry. Measurements requi...

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“…For the analysis of microvessel morphology, the parameters of the F-circle (4 9 area/p 9 max diameter 2 ) and the F-shape (min diameter/max diameter) were determined [10]. The detailed process was as follows: the full immunostained (Fig.…”

Section: Analysis Of Microvessel Density and Morphologymentioning

confidence: 99%

An immunohistochemical study of metroplastic surgical specimens from patients with a septate uterus

Makino

Izumi

et al. 2011

Reprod Medicine & Biology

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Purpose To elucidate the etiology of recurrent pregnancy loss in patients with congenital uterine anomalies, an immunohistochemical technique was used to quantitatively evaluate the vascular arrangement of septate uteri with respect to vascular density and morphology. Methods Nine specimens obtained from patients who had undergone metroplastic surgery for the treatment of a septate uterus and 10 control specimens from patients who had undergone a hysterectomy because of cervical carcinoma were used in this study. Formalin-fixed paraffinembedded uterine specimens were then immunostained for CD34, which is specifically expressed in vascular endothelial cells. Results The mean blood vessel count (mean ± SD) for the myometrium was 149.7 ± 22.7/field in the septate uteri and 162.2 ± 36.4/field in the control uteri; these values were not significantly different. However, the total vessel cross-sectional areas, as evaluated quantitatively using the KS400 image analysis system, were 10350.4 ± 1024.3 lm 2 /field for the septate uteri and 12002.9 ± 2232.3 lm 2 / field for the control uteri; these values were significantly different (p \ 0.05). The vessel morphology expressed by vessel irregularity and deformity showed a characteristic change in the septate uterus. Conclusions A significant difference in the distribution of the blood vessels existed between the septate and control uteri, presumably impairing blood flow in the myometrium and the adverse pregnancy outcome.

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An improved procedure to quantify tumour vascularity using true colour image analysis. Comparison with the manual hot-spot procedure in a human melanoma xenograft model

Cited by 43 publications

References 16 publications

The Angiopoietin-like Factor Cornea-derived Transcript 6 Is a Putative Morphogen for Human Cornea

The Angiopoietin-like Factor Cornea-derived Transcript 6 Is a Putative Morphogen for Human Cornea

Whole-body and intravital optical imaging of angiogenesis in orthotopically implanted tumors

An immunohistochemical study of metroplastic surgical specimens from patients with a septate uterus

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