In the three different stages of s.c. B16.F10 tumor growth, mRNA expression of the 46 genes under study is similar. Tumors were harvested at palpable, intermediate, and large volumes (early, intermediate and late stages). Values represent mRNA expression levels measured by qRT-PCR-based Low-Density Array and adjusted to GAPDH. Mean + SD; n = 3. Expression of FGF-2, FGFR2, Ang1, P-selectin, E-selectin, and COX-2 was additionally measured twice by manual qRT-PCR. PCNA was measured by manual real-time RT-PCR alone in duplicate. *P < 0.05. ND: not detectable.

Vascular compartmentalization of angio-gene expression in a large tumor. Tumor vasculature was laser microdissected and its gene expression profile was compared to that of the tumor as a whole. Values represent mRNA levels adjusted to GAPDH. Mean values ± SD of 3 qPCR analyses, except for CD31, VE-cadherin, vWF, EphrinB2, EphB4, Integrin αv, VEGFR2, Tie2, iNOS, eNOS, TNFα, TNFR1, TNFR2 (a total of 5 qPCR analyses), and for VEGF (a total of 7 qPCR analyses) and PCNA (qPCR in quadruplicate). *P < 0.05. ND: not detectable.

Vessels with different morphological appearance are covered with pericytes presenting with a different pattern of pericyte marker expression. (a, b) Immunofluorescent double staining for CD31 (green) and desmin (red) or α-SMA (red) and nuclear counter stain (DAPI; blue) of a representative tumor from the group of late-stage, large volume tumors (Magnification 200x). (c) Immunohistochemical detection of Ki67 revealed homogeneous distribution of proliferative activity throughout the whole tumor, in all three stages of tumor growth (Magnification 200x). Arrows point to Ki67-positive endothelial nuclei.

Morphological appearance of tumor vasculature of B16.F10 melanoma growing subcutaneously: immunohistochemical detection of CD31. Tumors were harvested at three different growth stages, based on tumor volumes as depicted in (e) (mean of 3 mice per group ± SEM). Vascular morphology differed per tumor growth stage, presenting vessels with increasing lumen size in intermediate and large tumors next to the presence of small vascular profiles, while small tumors were characterized by only small vessels that were generally lacking a visible lumen (Magnification 100x).

Vascular localization and expression of endothelial molecules at the protein level in B16.F10 tumors growing at three different stages of tumor growth. Expression of VCAM-1 and ICAM-1 was highest in the early-stage tumors, as compared to intermediate and large tumors. A granular pattern of vWF expression was found in a small number of tumor vessels; the majority of them were negative for vWF. Tie2 expression remained the same throughout tumor development and was observed in only a subset of the vessels. The top three and bottom three rows of panels represent immunohistochemical staining of consecutive sections. Arrowheads point to the structures that are enlarged in the insets. Thin arrows point to vessels in the large tumor that are positive for Tie2, while the largest vessel in the same photograph did not express detectable levels of Tie2 protein. Pictures are representative examples of each experimental group (Magnification 100x).

Different vascular morphologies displayed a similar pattern of gene expression. High variability of vascular morphology was observed in tumors of large volume. Large lumen-containing vessels ((b, d) vessel lumen size > 25 μm) existed next to much smaller blood vessels that did not contain a visible lumen (a, c). (a, b) Immunohistochemical staining for CD31: pictures represent different regions within the same large tumor (Magnification 100x). (c, d) Cryosections from large tumors were stained with GSL-I IsolectinB4:FITC and the different vascular morphologies were separately isolated by laser microdissection along the depicted lines, magnification 400x. Microdissection of these different vascular phenotypes followed by gene expression profiling did not reveal a differential pattern of mRNA expression of angiogenesis- and vascular stability-regulating genes (e), nor of the Notch family of genes that has recently been identified to be important regulators of both angiogenic sprouting and endothelial-pericyte adhesion (f), but demonstrated that the lumen-containing vessels were associated with significantly higher levels of αSMA mRNA compared to the small vessels without lumen (g). (e)–(g) Mean values + SD of duplicate qPCR measurements of three large tumors. ND: not detectable. *P < 0.05.

Summary of gain and loss of gene expression in the B16.F10 tumor vascular compartment of intermediate and large tumors compared to normal quiescent endothelium: gene expression in microdissected tumor endothelium from s.c. growing intermediate and large B16.F10 tumors (a total of 6 animals) and microdissected kidney venule endothelium from healthy C57bl/6 mice (10 animals). Red: detectable expression in more than 75% of all analyzed samples; yellow: detectable expression in 25–75% of all analyzed samples; green: expression of the gene could not be detected in the majority of the samples (less than 25% of positivity).