Abstract
Tumor hypoxia is commonly observed in primary solid malignancies but the hypoxic status of subclinical micrometastatic disease is largely unknown. The distribution of hypoxia in microscopic tumors was studied in animal models of disseminated peritoneal disease and intradermal (i.d.) growing tumors. Tumors derived from human colorectal adenocarcinoma cell lines HT29 and HCT-8 ranged in size from a few hundred microns to several millimeters in diameter. Hypoxia was detected by immunofluorescent visualization of pimonidazole and the hypoxia-regulated protein carbonic anhydrase 9. Tumor blood perfusion, cellular proliferation, and vascularity were visualized using Hoechst 33342, bromodeoxyuridine, and CD31 staining, respectively. In general, tumors of <1 mm diameter were intensely hypoxic, poorly perfused, and possessed little to no vasculature. Larger tumors (approximately 1-4 mm diameter) were well perfused with widespread vasculature and were not significantly hypoxic. Patterns of hypoxia in disseminated peritoneal tumors and i.d. tumors were similar. Levels of hypoxia in microscopic peritoneal tumors were reduced by carbogen breathing. Peritoneal and i.d. tumor models are suitable for studying hypoxia in microscopic tumors. If the patterns of tumor hypoxia in human patients are similar to those observed in these animal experiments, then the efficacy of systemic treatments of micrometastatic disease may be compromised by hypoxic resistance.
Highlights
The existence of tumor hypoxia is a common feature of primary solid malignancies [1,2,3,4,5], the hypoxic status of subclinical micrometastatic disease is largely unknown
We describe our study of the distribution of hypoxia in microscopic tumors using a disseminated peritoneal disease model and a focal intradermal (i.d.) model in athymic mice
The distribution of regions staining positive for pimonidazole and, for HT29 tumors, Carbonic anhydrase 9 (CA9) was examined in conjunction with tumor blood perfusion (Hoechst 33342) and morphology (H&E; Fig. 1A; Supplementary Fig. S1A–F)
Summary
The existence of tumor hypoxia is a common feature of primary solid malignancies [1,2,3,4,5], the hypoxic status of subclinical micrometastatic disease is largely unknown. One fundamental difficulty associated with characterizing microscopic disease is the inability to localize it spatially. This, together with the existence of multiple sites of dissemination, necessitates a systemic approach to treatment, typically using chemotherapeutic drugs. It follows that if hypoxic microscopic disease is present, it may be relatively resistant to treatment. The use of a serum hypoxia marker, such as osteopontin [8, 9], may enable the existence of hypoxic subclinical disease in patients to be inferred. In the presence of gross disease, the incremental ‘‘signal’’ from micrometastases is likely to be overwhelmed
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