Quantitative assessment of hypoxia subtypes in microcirculatory supply units of malignant tumors using (immuno-)fluorescence techniques.

BACKGROUND AND PURPOSE: Hypoxia is a characteristic of tumors, is known to increase aggressiveness, and causes treatment resistance. Traditional classification suggests two types of hypoxia: chronic and acute. Acute hypoxia is mostly caused by transient disruptions in perfusion, while chronic hypoxia is caused by diffusion limitations. This classification may be insufficient in terms of pathogenetic and pathophysiological mechanisms. Therefore, we quantified hypoxia subtypes in tumors based on (immuno-)fluorescent marker distribution patterns in microcirculatory supply units (MCSUs). MATERIAL AND METHODS: Cryosections from hSCC lines (SAS, FaDu, UT-SCC-5, UT-SCC-14, UT-SCC-15) were analyzed. Hypoxia was identified by pimonidazole, perfusion by Hoechst 33342, and endothelial cells by CD31. The following patterns were identified in vital tumor tissue: (1) normoxia: Hoechst 33342 fluorescence around microvessels, no pimonidazole, (2) chronic hypoxia: Hoechst 33342 fluorescence around microvessels, pimonidazole distant from microvessels, (3) acute hypoxia: no Hoechst 33342 fluorescence around microvessels, pimonidazole in immediate vicinity of microvessels, and (4) hypoxemic hypoxia: Hoechst 33342 fluorescence and pimonidazole directly around microvessels. RESULTS: Quantitative assessment of MCSUs show predominance for normoxia in 4 out of 5 tumor lines (50.1-72.8%). Total hypoxia slightly prevails in UT-SCC-15 (56.9%). Chronic hypoxia is the dominant subtype (65.4-85.9% of total hypoxia). Acute hypoxia only accounts for 12.9-29.8% and hypoxemic hypoxia for 1.2-6.4% of total hypoxia. The fraction of perfused microvessels ranged from 82.5-96.6%. CONCLUSION: Chronic hypoxia is the prevailing subtype in MCSUs. Acute hypoxia and hypoxemic hypoxia account for only a small fraction. This approach enables assessment and recognition of different hypoxia subtypes including hypoxemic hypoxia and may facilitate methods to (clinically) identify and eliminate hypoxia.

Preclinical evaluation of parametric image reconstruction of [18F]FMISO PET: correlation with ex vivo immunohistochemistry.

Compared to indirect methods, direct parametric image reconstruction (PIR) has the advantage of high quality and low statistical errors. However, it is not yet clear if this improvement in quality is beneficial for physiological quantification. This study aimed to evaluate direct PIR for the quantification of tumor hypoxia using the hypoxic fraction (HF) assessed from immunohistological data as a physiological reference. Sixteen mice with xenografted human squamous cell carcinomas were scanned with dynamic [18F]FMISO PET. Afterward, tumors were sliced and stained with H&E and the hypoxia marker pimonidazole. The hypoxic signal was segmented using k-means clustering and HF was specified as the ratio of the hypoxic area over the viable tumor area. The parametric Patlak slope images were obtained by indirect voxel-wise modeling on reconstructed images using filtered back projection and ordered-subset expectation maximization (OSEM) and by direct PIR (e.g., parametric-OSEM, POSEM). The mean and maximum Patlak slopes of the tumor area were investigated and compared with HF. POSEM resulted in generally higher correlations between slope and HF among the investigated methods. A strategy for the delineation of the hypoxic tumor volume based on thresholding parametric images at half maximum of the slope is recommended based on the results of this study.

Monitoring PAI-1 and VEGF levels in 6 human squamous cell carcinoma xenografts during fractionated irradiation.

PURPOSE: Previous studies have shown that the plasminogen activator inhibitor type-1 (PAI-1) and vascular endothelial growth factor (VEGF) are regulated by hypoxia and irradiation and are involved in neoangiogenesis. The aim of this study was to determine in vivo whether changes in PAI-1 and VEGF during fractionated irradiation could predict for radiation resistance. METHODS AND MATERIALS: Six xenografted tumor lines from human squamous cell carcinomas (HSCC) of the head and neck were irradiated with 0, 3, 5, 10, and 15 daily fractions of 2 Gy. The PAI-1 and VEGF antigen levels in tumor lysates were determined by enzyme-linked immunosorbent assay kits. The amounts of PAI-1 and VEGF were compared with the dose to cure 50% of tumors (TCD(50)). Colocalization of PAI-1, pimonidazole (hypoxia), CD31 (endothelium), and Hoechst 33342 (perfusion) was examined by immunofluorescence. RESULTS: Human PAI-1 and VEGF (hVEGF) expression levels were induced by fractionated irradiation in UT-SCC-15, UT-SCC-14, and UT-SCC-5 tumors, and mouse VEGF (msVEGF) was induced only in UT-SCC-5 tumors. High hVEGF levels were significantly associated with radiation sensitivity after 5 fractions (P=.021), and high msVEGF levels were significantly associated with radiation resistance after 10 fractions (P=.007). PAI-1 staining was observed in the extracellular matrix, the cytoplasm of fibroblast-like stroma cells, and individual tumor cells at all doses of irradiation. Colocalization studies showed PAI-1 staining close to microvessels. CONCLUSIONS: These results indicate that the concentration of tumor-specific and host-specific VEGF during fractionated irradiation could provide considerably divergent information for the outcome of radiation therapy.

Changes in the fraction of total hypoxia and hypoxia subtypes in human squamous cell carcinomas upon fractionated irradiation: evaluation using pattern recognition in microcirculatory supply units.

BACKGROUND AND PURPOSE: Evaluate changes in total hypoxia and hypoxia subtypes in vital tumor tissue of human head and neck squamous cell carcinomas (hHNSCC) upon fractionated irradiation. MATERIALS AND METHODS: Xenograft tumors were generated from 5 hHNSCC cell lines (UT-SCC-15, FaDu, SAS, UT-SCC-5 and UT-SCC-14). Hypoxia subtypes were quantified in cryosections based on (immuno-)fluorescent marker distribution patterns of Hoechst 33342 (perfusion), pimonidazole (hypoxia) and CD31 (endothelium) in microcirculatory supply units (MCSUs). Tumors were irradiated with 5 or 10 fractions of 2 Gy, 5×/week. RESULTS: Upon irradiation with 10 fractions, the overall fraction of hypoxic MCSUs decreased in UT-SCC-15, FaDu and SAS, remained the same in UT-SCC-5 and increased in UT-SCC-14. Decreases were observed in the proportion of chronically hypoxic MCSUs in UT-SCC-15, in the fraction of acutely hypoxic MCSUs in UT-SCC-15 and SAS, and in the percentage of hypoxemically hypoxic MCSUs in SAS tumors. After irradiation with 5 fractions, there were no significant changes in hypoxia subtypes. Changes in the overall fraction of hypoxic MCSUs were comparable to corresponding alterations in the proportions of acutely hypoxic MCSUs. There was no correlation between radiation resistance (TCD(50)) and any of the investigated hypoxic fractions upon fractionated irradiation. CONCLUSIONS: This study shows that there are large alterations in the fractions of hypoxia subtypes upon irradiation that can differ from changes in the overall fraction of hypoxic MCSUs.

Comparison of (immuno-)fluorescence data with serial [¹8F]Fmiso PET/CT imaging for assessment of chronic and acute hypoxia in head and neck cancers.

PURPOSE: Both, acute and chronic hypoxia can have unfavorable impacts on tumor progression and therapy response. The aim of this study was to optimize a macroscopic technique for the quantification of acute and chronic hypoxia (Wang model assessment of serial [(18)F]Fmiso PET/CT imaging) by comparing with a microscopic technique [(immuno-)fluorescence staining in tumor cryosections]. MATERIALS AND METHODS: Tumor pieces from the human squamous cell carcinoma lines from the head and neck FaDu and CAL33 were xenografted into the hind leg of NMRI nu/nu mice. Tumor-bearing mice were placed on an in-house developed multi-point fixation system and subjected to two consecutive dynamic [(18)F]Fmiso PET/CTs within a 24h interval. The Wang model was applied to SUV (standard uptake values) to quantify the fractions of acute and chronic hypoxia. Hypoxia subtypes were also assessed in vital tumor tissue of cryosections from the same tumors for (immuno-)fluorescence distributions of Hoechst 33342 (perfusion), pimonidazole (hypoxia), and CD31 (endothelium) using pattern recognition in microcirculatory supply units (defined as vital tumor tissue area supplied by a single microvessel). RESULTS: Using our multi-point fixation system, acceptable co-registration (registration errors ε ranged from 0.34 to 1.37) between serial PET/CT images within individual voxels was achieved. The Wang model consistently yielded higher fractions of acute hypoxia than the MCSU method. Through specific modification of the Wang model (Wang(mod)), it was possible to reduce the fraction of acute hypoxia. However, there was no significant correlation between the fractions of acute hypoxia in individual tumors assessed by the Wang(mod) model and the MCSU method for either tumor line (FaDu: r=0.68, p=0.21 and CAL33: r=0.71, p=0.18). This lack of correlation is most-likely due to the difference between the non-linear uptake of [(18)F]Fmiso and the spatial assessment of MCSUs. CONCLUSIONS: Whether the Wang model can be used to predict radiation response after serial [(18)F]Fmiso PET imaging, needs to be confirmed in experimental and clinical studies.