Abstract 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 FFmiso 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 FFmiso PET/CTs within a 24 h 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 (Wangmod ), 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 Wangmod 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 FFmiso and the spatial assessment of MCSUs. Conclusions Whether the Wang model can be used to predict radiation response after serial 18 FFmiso PET imaging, needs to be confirmed in experimental and clinical studies.
Purpose
Quantitative evaluation of tumor hypoxia based on
H
-1-(3-
18
Ffluoro-2-hydroxypropyl)-2-nitroimidazole (
18
FFMISO) positron emission tomography (PET) can deliver important information for ...treatment planning in radiotherapy. However, the merits and limitations of different analysis methods in revealing the underlying physiological feature are not clear. This study aimed to assess these quantitative analysis methods with the support of immunohistological data.
Procedures
Sixteen nude mice bearing xenografted human squamous cell carcinomas (FaDu or CAL-33) were scanned using 2-h dynamic
18
FFMISO PET. Tumors were resected and sliced, and the hypoxia marker pimonidazole was immunostained followed by H&E staining. The pimonidazole signal was segmented using a k-means clustering algorithm, and the hypoxic fraction (HF) was calculated as the hypoxic area/viable tumor-tissue-area ratio pooled over three tissue slices from the apical, center, and basal layers. PET images were analyzed using various methods including static analysis standard uptake value (SUV), tumor-to-blood ratio (T/B), tumor-to-muscle ratio (T/M) and kinetic modeling (Casciari
αk
A
, irreversible and reversible two-tissue compartment
k
3
, Thorwarth
w
A
k
3
, Patlak
K
i
, Logan
V
d
, Cho
K
), and correlated with HF.
Results
No significant correlation was found for static analysis. A significant correlation between
k
3
of the irreversible two-tissue compartment model and HF was observed (
r
= 0.61,
p
= 0.01). The correlation between HF and
αk
A
of the Casciari model could be improved through reducing local minima by testing more sets of initial values (
r
= 0.59,
p
= 0.02) or by reducing the model complexity by fixing three parameters (
r
= 0.63,
p
= 0.0008).
Conclusions
With support of immunohistochemistry data, this study shows that various analysis methods for
18
FFMISO PET perform differently for assessment of tumor hypoxia. A better fitting quality does not necessarily mean a higher physiological correlation. Hypoxia PET analysis needs to consider both the mathematical stability and physiological fidelity. Based on the results of this study, preference should be given to the irreversible two-tissue compartment model as well as the Casciari model with reduced parameters.
Positron-emission tomography (PET) with hypoxia specific tracers provides a noninvasive method to assess the tumor oxygenation status. Reaction-diffusion models have advantages in revealing the ...quantitative relation between in vivo imaging and the tumor microenvironment. However, there is no quantitative comparison of the simulation results with the real PET measurements yet. The lack of experimental support hampers further applications of computational simulation models. This study aims to compare the simulation results with a preclinical
FFMISO PET study and to optimize the reaction-diffusion model accordingly. Nude mice with xenografted human squamous cell carcinomas (CAL33) were investigated with a 2 h dynamic
FFMISO PET followed by immunofluorescence staining using the hypoxia marker pimonidazole and the endothelium marker CD 31. A large data pool of tumor time-activity curves (TAC) was simulated for each mouse by feeding the arterial input function (AIF) extracted from experiments into the model with different configurations of the tumor microenvironment. A measured TAC was considered to match a simulated TAC when the difference metric was below a certain, noise-dependent threshold. As an extension to the well-established Kelly model, a flow-limited oxygen-dependent (FLOD) model was developed to improve the matching between measurements and simulations. The matching rate between the simulated TACs of the Kelly model and the mouse PET data ranged from 0 to 28.1% (on average 9.8%). By modifying the Kelly model to an FLOD model, the matching rate between the simulation and the PET measurements could be improved to 41.2-84.8% (on average 64.4%). Using a simulation data pool and a matching strategy, we were able to compare the simulated temporal course of dynamic PET with in vivo measurements. By modifying the Kelly model to a FLOD model, the computational simulation was able to approach the dynamic
FFMISO measurements in the investigated tumors.
Background and Purpose:
Hypoxia is a characteristic of tumors, is known to increase aggressiveness, and causes treatment re-sistance. 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.
Ionizing irradiation is a commonly accepted treatment modality for lung cancer patients. However, the clinical outcome is hampered by normal tissue toxicity and tumor hypoxia. Since tumors often have ...higher levels of active heat shock protein 90 (Hsp90) than normal tissues, targeting of Hsp90 might provide a promising strategy to sensitize tumors towards irradiation. Hsp90 client proteins include oncogenic signaling proteins, cell cycle activators, growth factor receptors and hypoxia inducible factor-1alpha (HIF-1alpha). Overexpression of HIF-1alpha is assumed to promote malignant transformation and tumor progression and thus might reduce the accessibility to radiotherapy. Herein, we describe the effects of the novel Hsp90 inhibitor NVP-AUY922 and 17-allylamino-17-demethoxygeldanamycin (17-AAG), as a control, on HIF-1alpha levels and radiosensitivity of lung carcinoma cells under normoxic and hypoxic conditions. NVP-AUY922 exhibited a similar biological activity to that of 17-AAG, but at only 1/10 of the dose. As expected, both inhibitors reduced basal and hypoxia-induced HIF-1alpha levels in EPLC-272H lung carcinoma cells. However, despite a down-regulation of HIF-1alpha upon Hsp90 inhibition, sensitivity towards irradiation remained unaltered in EPLC-272H cells under normoxic and hypoxic conditions. In contrast, treatment of H1339 lung carcinoma cells with NVP-AUY922 and 17-AAG resulted in a significant up-regulation of their initially high HIF-1alpha levels and a concomitant increase in radiosensitivity. In summary, our data show a HIF-1alpha-independent radiosensitization of normoxic and hypoxic H1339 lung cancer cells by Hsp90 inhibition.
The objective of the study was to validate the model of empathic opportunity (EO) and potential empathic opportunity (PEO) using the Roter Interaction Analysis System (RIAS) in a sample of cancer ...patients. Thirty-nine audio taped consultations at an outpatient oncology clinic performed by four oncologists were previously coded with the Roter Interaction Analysis System for another purpose. These consultations were also coded by two raters with the empathic and potential empathic opportunity method (E-PE-O method). The reliability of EO and PEO coding was satisfactory. Most of the EOs were found within the RIAS category “showing concern”. The PEOs were found in both the socio-emotional and the instrumental categories of the RIAS. We conclude that the E-PE-O method is a good starting point for studying the empathy process in oncology consultations.
Many non-surgical tumor treatments induce reactive oxygen species (ROS) which result in cell damage. This study investigated the impact of ROS induction on the expression of adhesion molecules and ...whether α-tocopherol pre-treatment could have a protective effect. Experimental rat DS-sarcomas were treated with a combination of localized 44°C-hyperthermia, inspiratory hyperoxia and xanthine oxidase which together lead to a pronounced ROS induction. Further animals were pre-treated with α-tocopherol. The in vivoexpression of E- and N-cadherin, α-catenin, integrins αv, β3 and β5 as well as of the integrin dimer αvβ3 was assessed by flow cytometry. The expression of αv-, β3-integrin, of the αvβ3-integrin dimer and of E-cadherin was significantly reduced by the ROS-inducing treatment. This effect was partially reversible by α-tocopherol, indicating that ROS play a role in this process. N-cadherin, α-catenin and β5-integrin expression were unaffected by ROS. These results indicate that the expression of several adhesion molecules is markedly reduced by ROS and may result in a decrease in the structural stability of tumor tissue. Further studies are needed to clarify the impact of ROS induction on the metastatic behavior of tumors.
Several non-surgical tumor treatment modalities produce their cytotoxic activity by generating reactive oxygen species (ROS). Anti-oxidative enzymes such as superoxide dismutase (SOD) or exogenously ...supplied antioxidants may therefore reduce the efficacy of these treatments. The aim of the present study was to analyze the impact of (i) inhibiting SOD using 2-methoxyestradiol (2-ME), or (ii) application of α-tocopherol, on the cellular damage induced by hyperthermia (HT) in experimental tumors. DS-sarcoma cells grew either in culture or as solid tumors subcutaneously implanted in rats. In vitro, DS-cells were incubated with 2-ME, and cell proliferation, ROS formation, lipid peroxidation and apoptosis were measured. In vivo, DS-sarcomas were treated with a ROS-generating hyperthermia combined with 2-ME or α-tocopherol application.
Inhibition of SOD by 2-ME in vitro induced pronounced oxidative injury resulting in reduced proliferation. In vivo, ROS-generating hyperthermia led to local tumor control in 23% of the animals. The additional inhibition of SOD by 2-ME increased the control rate by approximately 50%. Application of α-tocopherol was found to have no effect on local tumor control, either in combination with ROS-generating hyperthermia or when 2-ME was additionally applied. Inhibition of SOD during ROS-generating hyperthermia results in pronounced cell injury and an improved local tumor control whereas exogenously applied vitamin E seems not to have an impact on oxidative stress.
Although strong evidence is available suggesting that microenvironmental parameters play a role in lymphogenic or hematogenic metastasis, the underlying mechanisms are still unclear and further ...investigations of this topic are needed. For such a study however, an appropriate model of metastasis for in vivo analysis of this process would be required. An in vivo model of a solid tumor (rat DS sarcoma) has therefore been established to enable monitoring of the steps involved in tumor metastasis. Rat DS sarcoma cells were transfected with the pTracer-SV40 plasmid, containing the super-GFP and zeocin resistance genes. DS sarcoma cells showing high and stable expression of GFP (DSGFP cells) were selected by cell sorting and in vitro culturing with zeocin. To establish in vivo growth, DSGFP cells were subsequently injected intraperitoneally (i.p.) without additional selection by zeocin and GFP expression was monitored by flow cytometry. Using DSGFP ascites cells, solid tumors were implanted subcutaneously into the hind foot dorsum of rats. The expression of GFP was assayed by fluorescence microscopy. The detection of circulating DSGFP sarcoma cells in the blood was performed using the PCR technique. GFP expression in vitro was stable for more than 40 passages. Cell sorting, however, did not enable selection of a DSGFP cell population with a higher long-term stable GFP expression. After i.p. cell implantation, GFP expression in DSGFP ascites cells was maintained over at least 19 passages. Solid tumors implanted by injection of DSGFP ascites cells showed stable GFP expression. The growth rate of solid DSGFP sarcomas was slightly slower compared to that of non-transfected cell lines. The detection limit for circulating DS sarcoma cells in blood was 100 DSGFP cells/ml whole rat blood. Micrometastases in loco-regional lymph nodes, lung and liver were detectable by immunohistology and real-time PCR. This in vivo model showing stable expression of GFP could be useful for analyzing the mechanisms of metastasis, particularly where micrometastases or circulating tumor cells are to be identified.
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