Vascular phenotype in angiogenic and non-angiogenic lung non-small cell carcinomas

Abstract

We have previously described a group of non-small cell lung carcinomas without morphological evidence of neo-angiogenesis. In these tumours neoplastic cells fill up the alveoli and the only vessels present appear to belong to the trapped alveolar septa. In the present study we have characterised the phenotype of the vessels present in these non-angiogenic tumours, in normal lung and in angiogenic non-small cell lung carcinomas. The vessels, identified by the expression of CD31, were scored as mature when expressing the epitope LH39 in the basal membrane and as newly formed when expressing alphaVbeta3 on the endothelial cells and/or lacking LH39 expression. In the nine putative non-angiogenic cases examined, the vascular phenotype of all the vessels was the same as that of alveolar vessels in normal lung: LH39 positive and alphaVbeta3 variable or negative. Instead in 104 angiogenic tumours examined, only a minority of vessels (mean 13.1%; range 0--60%) expressed LH39, while alphaVbeta3 (in 45 cases) was strongly expressed on many vessels (mean 55.5%; range 5--90%). We conclude that in putative non-angiogenic tumours the vascular phenotype is that of normal vessels and there is no neo-angiogenesis. This type of cancer may be resistant to some anti-angiogenic therapy and different strategies need to be developed.

Figure 1

Normal lung (frozen sections). (A) (×400) Double immunostaining for LH39 (in brown) and CD31 (in blue); no counterstaining was done. The vessels of the alveolar walls have LH39 expression in their membrane. (B) (×400) Staining for αVβ3 (in brown). The nuclei are counter stained with haematoxilin (blue). A variable positivity on the endothelial cells is present.

Figure 2

A non-angiogenic tumour (frozen sections). (A) (×100) (low magnification) and (B) (×400) double immunostaining for LH39 (in brown) and CD31 (in blue); no counterstaining was done. All the vessels express LH39 in the basal membrane. (C) (×100), (D) (×400) Immunostaining for αVβ3 (in brown); the nuclei are counter stained with haematoxilin (blue). A variable positivity on the endothelial cells is present as seen in normal lung (Figure 1).

Figure 3

An angiogenic lung carcinoma (frozen sections). (A) (×400) double immunostaining for LH39 (in brown) and CD31 (in blue) no counter staining was done. Only a few vessels express LH39 in the basal membrane. (B) (×400) Immunostaining for αVβ3 (in brown). The nuclei are counter stained with haematoxilin (blue). Some vessels are strongly stained.

Figure 4

Phenotype of intratumour vessels in 44 angiogenic lung n-SCLC. Three groups of tumours can be identified. Groups A (diamonds) and B (triangles) are made up of a mixture of mature (LH39 positive) and immature (LH39 negative) vessels. However while in group A all the immature express αVβ3, in group B the immature are, in part, αVβ3 negative. Group C (circles) is made up almost completely by new vessels (LH39 negative) but only a proportion are αVβ3 positive. N.B.: each symbol in the diagram may well represent more than one sample.

Figure 5

Angiogenic tumour, basal pattern with residual non angiogenic component (frozen sections). Double immunostaining for LH39 (in brown) and CD31 (in blue), no counter staining was done. (A) (×100) In some areas an alveolar pattern is still present. The arrows indicates where tumour-associated stroma is present and where remodelling and angiogenesis start. (B) (×400) Islands of neoplastic cells survive surrounded by stroma with new vessels.

Figure 6

Early papillary pattern (frozen sections). (A) (×40) Double immunostaining for LH39 (in brown) and CD31 (in blue). No counter staining was done. The alveolar pattern appears preserved throughout the tumour. (B) (×100) Haematoxilin and Eosin (C) (×100) CD31 (in brown, the nuclei are counter stained with haematoxilin (blue)) and (D) (×100) CD34 (in brown, the nuclei are counter stained with haematoxilin (blue)). Within these micropapillae a small vessel (arrow), positive for CD31 and CD34, is present. (E) (×100) Double immunostaining for LH39 (in brown) and CD31 (in blue). No counter staining was done. A new vessel CD31 positive but LH39 negative (arrow) is present in the stalk of this micropapilla. (F) (×100) Staining for αVβ3 (in brown). The nuclei are counter stained with haematoxilin (blue). An αVβ3 positive microvessel (arrow) within an early papilla.