Experimental imagingImaging of CA IX with fluorescent labelled sulfonamides distinguishes hypoxic and (re)-oxygenated cells in a xenograft tumour model
Section snippets
Cell culture, animal and tumour model
Exponentially growing colorectal (HT-29, ATCC HTB-38) carcinoma cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. Hypoxic conditions were acquired as described previously [12]. In parallel, normoxic dishes were incubated in air with 5% CO2. Animal experiments were performed using adult NMRI-nu (nu/nu) female mice with an average body weight of 30 g. The animal facilities and experiments were in accordance with local institutional guidelines for
Results
To mimic the tumour micro-environment, HT-29 cells were exposed to normoxic (24 h), hypoxic (24 h 0.2%) or reoxygenation (24 h hypoxia 0.2%, followed by 1 h normoxia) conditions, and were incubated with CAI(+) for the last 30 min of the different exposures. Cells were stained with nuclear markers to identify hypoxic or (re)-oxygenated groups, mixed and analyzed. Flow cytometry demonstrated a significant higher CAI(+) accumulation (P < 0.01) in hypoxic (Syto 41 gated) cells, compared with their
Discussion
Fluorescent labelled sulfonamides with high affinity for CA IX can be used to visualize hypoxic HT-29 cells characterized by active CA IX. CAI(+) accumulation correlated with a strong CA IX protein upregulation in HT-29 cells exposed to hypoxia. HT-29 cells also demonstrated intermediate CA IX expression under normoxic conditions, probably due to cell density-related CA IX expression [18] and CA IX levels remained elevated upon reoxygenation, in agreement with the known CA IX half-life of 38 h
Conclusion
Sulfonamides with high affinity for CA IX specifically accumulate in delineated regions of human xenografts. This accumulation is dependent on the presence of hypoxia and is reversible upon tumour reoxygenation, providing a tool to image hypoxia response. Furthermore, our data indicate the potential of patient selection for hypoxia-directed and/or CA IX-directed therapies.
Acknowledgements
This work has been funded with the support of the EU 6th framework Program (Euroxy Project Ref. 2003-502932), the EU 7th framework (Metoxia Project Ref. 2008-222741) and the Dutch Cancer Society (KWF Grant UM 2008-4068).
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