Elsevier

Radiotherapy and Oncology

Volume 92, Issue 3, September 2009, Pages 423-428
Radiotherapy and Oncology

Experimental imaging
Imaging of CA IX with fluorescent labelled sulfonamides distinguishes hypoxic and (re)-oxygenated cells in a xenograft tumour model

https://doi.org/10.1016/j.radonc.2009.06.019Get rights and content

Abstract

Background and purpose

Carbonic anhydrase (CA) IX is suggested to be an endogenous marker of hypoxia. Fluorescent sulfonamides with a high affinity for CA IX (CAI) have been developed and shown to bind to cells only when CA IX protein was expressed and while cells were hypoxic. The aim of this study was to investigate the in vivo CAI binding properties in a xenograft tumour model using fluorescent imaging.

Materials and methods

NMRI-nu mice subcutaneously transplanted with HT-29 colorectal tumours were treated with 7% oxygen or with nicotinamide and carbogen and were compared with control animals. CAI accumulation was monitored by non-invasive fluorescent imaging.

Results

Specific CAI accumulation could be observed in delineated tumour areas as compared with a non-sulfonamide analogue (P < 0.01). Administration of nicotinamide and carbogen, decreasing acute and chronic hypoxia, respectively, prevented CAI accumulation (P < 0.05). When treated with 7% oxygen breathing, a 3-fold higher CAI accumulation (P < 0.01) was observed. Furthermore, the bound CAI fraction was rapidly reduced upon tumour reoxygenation (P < 0.01).

Conclusions

Our in vivo imaging results confirm previous in vitro data demonstrating that CAI binding and retention require exposure to hypoxia. Fluorescent labelled sulfonamides provide a powerful tool to visualize hypoxia response. An important step is made towards clinical applicability, indicating the potential of patient selection for CA IX-directed therapies.

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).

References (27)

  • J. Bussink et al.

    Vascular architecture and microenvironmental parameters in human squamous cell carcinoma xenografts: effects of carbogen and nicotinamide

    Radiother Oncol

    (1999)
  • E.G. Troost et al.

    Comparison of different methods of CAIX quantification in relation to hypoxia in three human head and neck tumor lines

    Radiother Oncol

    (2005)
  • J.M. Brown et al.

    The unique physiology of solid tumors: opportunities (and problems) for cancer therapy

    Cancer Res

    (1998)
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