Quantitative Imaging of the Hypoxia-Related Marker CAIX in Head and Neck Squamous Cell Carcinoma Xenograft Models

Abstract

Tumor hypoxia plays a major role in radio- and chemotherapy resistance in solid tumors. Carbonic Anhydrase IX (CAIX) is an endogenous hypoxia-related protein, which is associated with poor patient outcome. The quantitative assessment of CAIX expression of tumors may steer cancer treatment by predicting therapy response or patient selection for antihypoxia or CAIX-targeted treatment. Recently, the single-photon emission computerized tomography (SPECT) tracer [111In]In-DTPA-girentuximab-F(ab′)2 was developed and validated for targeting CAIX. The aim of this study was to optimize quantitative microSPECT/CT of CAIX expression in vivo in head and neck tumor models. Athymic mice with subcutaneous SCCNij153 and SCCNij202 head and neck squamous cell carcinoma xenografts were injected with [111In]In-DTPA-girentuximab-F(ab′)2. First, the protein dose, timing, and image acquisition settings were optimized. Tracer uptake was determined by quantitative SPECT, ex vivo radioactivity counting, and by autoradiography of tumor sections. The same tumor sections were immunohistochemically stained for CAIX expression and hypoxia. Highest tumor-normal-tissue contrast was obtained at 24 h after injection of the tracer. A protein dose of 10 μg resulted in the highest tumor-to-muscle ratio at 24 h p.i. Ex vivo biodistribution studies showed a tumor uptake of 3.0 ± 0.6%ID/g and a tumor-to-muscle ratio of 8.7 ± 1.4 (SCCNij153). Quantitative analysis of the SPECT images enabled us to distinguish CAIX antigen blocked from nonblocked tumors, fractions positive for CAIX expression: 0.22 ± 0.02 versus 0.08 ± 0.01 (p < 0.01). Immunohistochemical, autoradiographic, and microSPECT/CT analyses showed a distinct intratumoral spatial correlation between localization of the radiotracer and CAIX expression. Here, we demonstrate that [111In]In-DTPA-girentuximab-F(ab′)2 specifically targets CAIX-expressing cells in head and neck cancer xenografts. SPECT imaging with indium-labeled girentuximab-F(ab′)2 allows quantitative assessment of the fraction of CAIX positive tissue in head and neck cancer xenografts. These results indicate that [111In]In-DTPA-girentuximab-F(ab′)2 is a promising tracer to image hypoxia-related CAIX expression.

Comparative evaluation of affibody- and antibody fragments-based CAIX imaging probes in mice bearing renal cell carcinoma xenografts

“…The use of [ 111 In]In-DTPA-G250(Fab’) 2 demonstrated earlier excellent potential for imaging of hypoxia-induced CAIX-expression in head-and-neck cancer 32,48 . This construct is appreciably larger (110 kDa) than the affibody molecules (7–8 kDa), and its clearance from blood and other tissues is slower (Table 1).…”

“…DTPA-G250(Fab’) 2 was produced by enzymatic digestion of the monoclonal chimeric anti-CAIX antibody girentuximab (G250 Wilex AG) with pepsine and conjugated with isothiocyanatobenzyl-diethylenetriaminepentaacetic acid (ITC-DTPA, Macrocyclis,Houston, TX, USA) as described previously 32,48 .…”

“…Labeling of DTPA-G250(Fab’) 2 with 111 In was performed as described and validated earlier 32,48 . Briefly, a stock solution of 40 μg DTPA-G250(Fab’) 2 (60 μL) was diluted in 120 μL 0.5 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer, pH 5.4, and mixed with 60 μL (20 MBq) [ 111 In]InCl 3 .…”

CAIX-targeting radiotracers for hypoxia imaging in head and neck cancer models

“…multiple CAIX-targeting tracers with very high tumor uptake levels and tumor-to-muscle ratios were discussed 20 . The highest ratios were found in experiments in mice with ccRCC xenografts, but these tracers were not tested in a hypoxia driven model 19,24–28 . One of the remaining questions is which tracer is most promising for the discrimination of low CAIX expressing tumors from high CAIX expressing tumors.…”

“…These fragments show a much faster blood clearance, but also a lower absolute tumor tracer uptake. Successful imaging with radiolabeled girentuximab-F(ab′) 2 is possible as early as 24 hours post tracer injection 18,19 . In recent years other approaches have proven to be successful as well 20 , including application of engineered molecules, called affibody molecules.…”