Hypoxia is an important feature of advanced head and neck tumors, with a negative influence on prognosis[12, 20]. Evaluation of cellular responses to hypoxia can be of clinical relevance in a prognostic and predictive way and possibly for treatment adaptation. Until now no endogenous marker has been found that strongly and consistently correlates with hypoxia, so different kind of analyses as presented here are crucial to elucidate the role of the various proteins and the response of tumor cells to a hypoxic microenvironment.
Although a global analysis of the overall expression of a certain protein allows easy comparison between biopsies and between different studies, it is a huge simplification of the true heterogeneous situation in a tumor biopsy. Therefore, to gain more insight in the spatial relationship between the metabolic markers and the oxygenation status in the tumor, we additionally assessed the expression of the proteins within and outside pimonidazole stained areas and the relation to the vessels. With this analysis more markers show an evident relationship with pimonidazole than with a global correlation analysis (Figure 4).
Of the endogenous hypoxia-related markers that we examined, LDH-5 has the strongest relationship with pimonidazole. LDH-5 is one of the target enzymes of HIF-1 and has been described to have a strong association with HIF-1α expression in tumor tissue sections [21, 22]. The high expression of LDH-5 in hypoxic areas is as expected; it is one of the key enzymes in the glycolysis, the primary energy source in the absence of oxygen. The reason for a considerable expression of LDH-5 outside hypoxic areas could be the high rate of aerobe glycolysis (the Warburg effect) as often observed in malignant tumors, although alternative explanations are possible, as pyruvate can originate from other pathways as well. It should be noted that due to the similarity between LDH-5 and the other LDH isoforms, there is a possibility of some cross-reactivity of the antibody.
CAIX is previously described to correlate weakly with pimonidazole binding[13, 24, 25]. Despite the weak overall correlation, CAIX fraction was significantly higher in the hypoxic (pimonidazole positive) areas. This emphasizes the strong association of this protein with hypoxia that could not be found by a simple correlation analysis. Interestingly, although all the endogenous markers, with the exception of MCT1, are regulated by HIF-1[9, 10], the staining patterns and colocalization differ. An interesting observation is the strong resemblance of the HIF-1α and CAIX staining pattern (intermediate hypoxic areas). This typical pattern circumferences the pimonidazole stained area with a partial overlap (Figure 2i). The colocalization of CAIX with HIF-1α is far from perfect, but more concordant than that of GLUT-1, MCT4 and LDH-5 with HIF-1α. The strong relationship of CAIX with HIF-1 is known, with CAIX expression tightly controlled by HIF-1. In this context, an interesting observation is the higher level of HIF-1α expression in hypoxia-related CAIX areas than in CAIX areas separate from hypoxic (pimonidazole stained) areas (Figure 2i/j). A possible explanation of these findings could be the increased transcription of HIF-1α without stabilization in the intermediate hypoxic areas (areas with CAIX expression without HIF-1α staining) and stabilization of HIF-1α in severe hypoxic areas (areas overlapping and adjacent to pimonidazole with CAIX and HIF-1α staining). It could also indicate the transient presence of acute hypoxic areas, which are reoxygenated before pimonidazole administration. It was shown in SiHa (human cervical squamous cell carcinoma) tumors that up to 20% of the tumor cells were intermittently hypoxic over an 8-hour period. This explanation is less likely as CAIX upregulation requires a longer period of hypoxia. A third explanation could be the induction of HIF-1α and consequent activation of CAIX through a hypoxia-independent mechanism without stabilization of HIF-1α under these conditions. An acidic tumor microenvironment can influence HIF-1α and CAIX expression as well[29, 30]. In any case, there is little evidence for a HIF-1 independent CAIX activating mechanism.
Most markers showed a range of co-expression, but a very strong spatial relationship was found between GLUT-1 and MCT4 (Figure 4h). These showed a comparable, typical expression increasing at larger distances from the vessels, with a two- to four-fold increase of the fraction at 150-200 μm. This pattern and agreement reflects their interrelated role in the glycolytic pathway under hypoxic conditions, GLUT-1 for glucose import and MCT4 for lactate export. LDH-5, one of the intermediary enzymes in this pathway, shows a similar pattern, but was not stained in the same section as MCT4 or GLUT-1, so the amount of colocalization could not be calculated.
Although a small series, a positive correlation between MCT4 and N-stage was found. Overexpression of MCT4 in malignancies has been described in colorectal cancer and cervical carcinomas. Except for a trend towards shorter overall survival with MCT4 positive adenosquamous carcinomas of the cervix, no correlations with clinicopathological data have been found before. These findings indicate that MCT4 is a potential marker for the aggressiveness of a tumor.
MCT1 expression in > 5% of the tumor area was present in 14 of the 20 biopsies. MCT1 expression in tumors has been described in lung cancer, brain tumors and cervical cancer. However, in the colonic epithelium, Lambert et al. found a decline in expression associated with transition to malignancy. In our study, MCT1 expression was present in oxic as well as hypoxic areas, in contrast to the observation made by Sonveaux et al. in biopsies of lung carcinomas. It is important to note that in our study a different antibody was used of which some aspecificity formally can not be excluded, although other studies show good results with this antibody as well[7, 36]. As the fraction MCT1 even increased at larger distance from the vessels, it seems likely that MCT1, beside MCT4, plays a role in lactate export in hypoxic areas. It is reasonable to assume that in tumor cells, like in red blood cells, depending on the substrate concentrations and pH, MCT1 functions as either a lactate importer or assists MCT4 in lactate export. Either way, MCT1 has already shown some potential as a therapeutic target in vitro. Inhibition of MCT1 by lonidamine induced a strong decrease in intracellular pH and loss of viability of the tumor cells. The MCT1 inhibitor α-cyano-4-hydroxycinnamate blocks lactate-fueled respiration in tumor cells and induces tumor growth retardation in a mouse model. Of interest is the strong correlation and colocalization of MCT1 with CAIX (Figure 3 and 4e), affirming an important role in pH regulation as described in melanoma and neuroblastoma[34, 38].
In conclusion, metabolic markers show a strong but irregular relation with hypoxia with obvious correlations between markers, emphasizing the complex metabolic regulatory system with a strong environmental (hypoxia, pH) influence. Co-expression of markers provides additional information over single marker fractions. MCT4 and GLUT-1 show a typical "diffusion-limited hypoxic" pattern with a strong colocalization indicating activation by similar stimuli. The positive correlation with N-stage makes MCT4 a potential marker for tumor aggressiveness, but its exact value still has to be established. MCT1 overexpression is present in the majority of the advanced head and neck carcinomas in our series, in non-hypoxic as well as hypoxic areas. The strong colocalization with CAIX suggests an important role in pH regulation.