Metabolic markers in relation to hypoxia; staining patterns and colocalization of pimonidazole, HIF-1α, CAIX, LDH-5, GLUT-1, MCT1 and MCT4

Abstract

Background: The cellular response of malignant tumors to hypoxia is diverse. Several important endogenous metabolic markers are upregulated under hypoxic conditions. We examined the staining patterns and co-expression of HIF-1α, CAIX, LDH-5, GLUT-1, MCT1 and MCT4 with the exogenous hypoxic cell marker pimonidazole and the association of marker expression with clinicopathological characteristics.

Methods: 20 biopsies of advanced head and neck carcinomas were immunohistochemically stained and analyzed. All patients were given the hypoxia marker pimonidazole intravenously 2 h prior to biopsy taking. The tumor area positive for each marker, the colocalization of the different markers and the distribution of the markers in relation to the blood vessels were assessed by semiautomatic quantitative analysis.

Results: MCT1 staining was present in hypoxic (pimonidazole stained) as well as non-hypoxic areas in almost equal amounts. MCT1 expression showed a significant overall correlation (r = 0.75, p < 0.001) and strong spatial relationship with CAIX. LDH-5 showed the strongest correlation with pimonidazole (r = 0.66, p = 0.002). MCT4 and GLUT-1 demonstrated a typical diffusion-limited hypoxic pattern and showed a high degree of colocalization. Both MCT4 and CAIX showed a higher expression in the primary tumor in node positive patients (p = 0.09 both).

Conclusions: Colocalization and staining patterns of metabolic and hypoxia-related proteins provides valuable additional information over single protein analyses and can improve the understanding of their functions and environmental influences.

Figure 1

MCT4 and CAIX expression according to nodal stage. A significantly higher expression of MCT4 (p = 0.04, a) and CAIX (p = 0.05, b) was found in node-positive tumours. One tumor without nodal metastasis had a high CAIX expression of 17%. Interestingly, no HIF-1α expression was found in this tumor.

Figure 2

Immunofluorescent staining pattern of metabolic and hypoxic markers. (a and b) Fluorescent microscopic images of two head and neck carcinomas with different pimonidazole (green) and CAIX (blue) staining patterns. (c - h) Examples of staining of the various markers. (c) LDH-5 (red), pimonidazole (green) and vessels (white). (d) Perinecrotic pimonidazole (green) and CAIX (red) staining, N = Necrosis. (e) MCT1 (red), pimonidazole (green) and vessels (white), note the co-staining of MCT1 with pimonidazole. (f and g) Membranous GLUT-1 (f) and MCT4 (g) (both red) staining in relation to the vessels (white). (h) Nuclear HIF-1α (red) expression in relation to pimoniazole (green) staining. (i and j) Fluorescent microscopic images showing intense HIF-1α (red) staining in a pimonidazole-related (green) CAIX (blue) area and almost absent HIF-1α staining in another CAIX area in the same tissue section.

Figure 3

Correlations between the endogenous markers and pimonidazole. A schematic representation has been added; solid lines represent a significant correlation, dashed lines a trend (p-value between 0.05-0.1). Percentages in the second table are mean values.

Figure 4

Colocalization of the various markers. (a-d) CAIX, LDH-5 and MCT4 show a significantly higher expression in pimonidazole stained areas, MCT1 expression is not significantly different. (e and f) Expression of MCT1 and MCT4 in CAIX negative and positive areas, both are significantly higher in areas expressing CAIX. (g and h) Increased expression of MCT1 and GLUT in MCT4 positive areas.

Figure 5

Marker expression in different zones around the vessels. Zone analysis in a biopsy with a large hypoxic fraction (upper panels) and a tumor with no pimonidazole staining (lower panels). Pimonidazole staining increases steadily at larger distances from the vessels (a). The increase of GLUT, MCT4 and LDH-5 expression is less steep with a plateau reached between 150-200 μm (b). MCT1 expression increases until 150-200 μm, a slight decrease is noted at larger distances (a). In the tumour with no pimonidazole staining, a decrease in MCT1 expression (c) and no increase of GLUT, MCT4 and LDH-5 expression (d) is observed at larger distances from the vessels.