The use of contrast-enhanced MRI to enable in vivo specific characterization of atherosclerotic p... more The use of contrast-enhanced MRI to enable in vivo specific characterization of atherosclerotic plaques is increasing. In this study the intrinsic ability of two differently sized gadolinium-based contrast agents to enhance atherosclerotic plaques in ApoE(-/-) mice was evaluated with MRI. We obtained a kinetic profile for contrast enhancement, as the literature data on optimal imaging time points is scarce, and assessed the longer-term kinetics. Signal enhancement in the wall of the aortic arch, following intravenous injection of paramagnetic micelles and liposomes, was followed for 1 week. In vivo T(1)-weighted MRI plaque enhancement characteristics were complemented by fluorescence microscopy of NIR(664) incorporated in the contrast agents and quantification of tissue and blood Gd-DTPA. Both micelles and liposomes enhanced contrast in T(1)-weighted MR images of plaques in the aortic arch. The average contrast-to-noise ratio increased after liposome or micelle injection to 260 or 2...
This paper presents a fully automated method for atlas-based whole-body segmentation in non-contr... more This paper presents a fully automated method for atlas-based whole-body segmentation in non-contrastenhanced Micro-CT data of mice. The position and posture of mice in such studies may vary to a large extent, complicating data comparison in cross-sectional and follow-up studies. Moreover, Micro-CT typically yields only poor soft-tissue contrast for abdominal organs. To overcome these challenges, we propose a method that divides the problem into an atlas constrained registration based on high-contrast organs in Micro-CT (skeleton, lungs and skin), and a soft tissue approximation step for low-contrast organs. We first present a modification of the MOBY mouse atlas (Segars et al., 2004) by partitioning the skeleton into individual bones, by adding anatomically realistic joint types and by defining a hierarchical atlas tree description. The individual bones as well as the lungs of this adapted MOBY atlas are then registered one by one traversing the model tree hierarchy. To this end, we employ the Iterative Closest Point method and constrain the Degrees of Freedom of the local registration, dependent on the joint type and motion range. This atlas-based strategy renders the method highly robust to exceptionally large postural differences among scans and to moderate pathological bone deformations. The skin of the torso is registered by employing a novel method for matching distributions of geodesic distances locally, constrained by the registered skeleton. Because of the absence of image contrast between abdominal organs, they are interpolated from the atlas to the subject domain using Thin-Plate-Spline approximation, defined by correspondences on the already established registration of high-contrast structures (bones, lungs and skin). We extensively evaluate the proposed registration method, using 26 non-contrast-enhanced Micro-CT datasets of mice, and the skin registration and organ interpolation, using contrast-enhanced Micro-CT datasets of 15 mice. The posture and shape varied significantly among the animals and the data was acquired in vivo. After registration, the mean Euclidean distance was less than two voxel dimensions for the skeleton and the lungs respectively and less than one voxel dimension for the skin. Dice coefficients of volume overlap between manually segmented and interpolated skeleton and organs vary between 0.47 ± 0.08 for the kidneys and 0.73 ± 0.04 for the brain. These experiments demonstrate the method's effectiveness for overcoming exceptionally large variations in posture, yielding acceptable approximation accuracy even in the absence of soft-tissue contrast in in vivo Micro-CT data without requiring user initialization.
Breast cancer frequently metastasizes to bone, where tumor cells induce osteoclasts to locally de... more Breast cancer frequently metastasizes to bone, where tumor cells induce osteoclasts to locally destroy bone. During bone resorption, growth factors are locally released that may support bone metastatic growth. Differently from most other tissues, drugs that can limit local turnover, such as bisphosphonates and osteoprotegerin (OPG), are available for bone. We examined the hypothesis that inhibition of bone resorption by two different mechanisms may also affect the growth of cancer cells in bone. For this, we tested the effects of high doses of OPG and zoledronic acid (ZOL) on progression of MDA-231-B/Luc+ breast cancer cells in the bone microenvironment using whole body bioluminescent reporter imaging (BLI). Both treatments significantly inhibited the development of radiographically detectable osteolytic lesions. Histologic examination corroborated the radiographic findings, showing that both treatments preserved the integrity of bone trabeculae and prevented bone destruction (significantly higher trabecular bone volumes vs. vehicle). However, whereas practically no TRAcP-positive osteoclasts were observed in tibiae preparations of animals treated with Fc-OPG, TRAcP-positive osteoclasts were still present in the animals treated with ZOL. Intra-bone tumor burden was reduced with ZOL and Fc-OPG treatment. Although there appeared to be a trend for less overall total tumor burden upon treatment with both compounds, this was not significant as assessed by BLI and histomorphometric analysis due to the extramedullary growth of cancer cells which was not affected by these treatments. Collectively, anti-resorptive agents with different mechanisms of action-ZOL and OPGsignificantly reduced cancer-induced osteolysis and intra-osseous tumor burden, but failed to restrain local tumor growth. However, interference with the bone micro-environmental growth support could still be of therapeutic relevance when given to patients early in the course of bone metastatic disease.
Prolonged 17-estradiol (E2) administration affects insulin sensitivity. However, it is unknown wh... more Prolonged 17-estradiol (E2) administration affects insulin sensitivity. However, it is unknown whether E2 influences insulin sensitivity directly or indirectly e.g. via modulation of plasma free fatty acid levels and/or intra-hepatic lipid levels. Therefore, acute effects of E2 administration were studied by performing a hyperinsulinemic-euglycemic clamp in an insulin resistant mouse model (APOE*3-Leiden mice, which had been fed a high fat diet for 13 weeks). Six hours after E2 administration, estrogen receptor mediated transcription was induced predominantly in liver, but plasma triglyceride, insulin, free fatty acid and intrahepatic lipid levels were unaffected. During the hyperinsulinemic clamp, the hepatic glucose production was significantly inhibited in the E2 treated mice as compared to control mice (4.5 11 versus 34 29 mol·min·kg; P=0.013), whereas the peripheral glucose disposal rate was significantly lower in the E2 treated mice (29.8±8.9 versus 50.9±26.4 mol·min·kg ; P=0....
aligned to the human reference genome (UCSC hg19) to generate the transcriptome. Differential exp... more aligned to the human reference genome (UCSC hg19) to generate the transcriptome. Differential expression analysis between HE-1100 and placebo was made in R using the DESeq2 package to identify the differentially expressed genes in the OA-associated regulatory pathways. The protein production of the selected genes was quantified by ELISA in 10 independent human OA chondrocytes cultures. Results: According to the DESeq2 analysis, HE-1100 significantly modified the expression of 13 genes in OA chondrocytes by at least 10% with an adjusted p-value < 0.05 : EGR1 (þ93%), FOS (þ87%), NR4A1 (þ43%), DUSP1 (þ18%), ZFP36 (þ18%), ZFP36L1 (þ14%), NFKBIZ (þ16%) and CYR61 (þ14%) were upregulated and ATF7IP (-10%), TXNIP (-11%), C10orf10 (-12%), CLEC3A (-12%) and MMP13 (-18%) were downregulated after 24h HE-1100 treatment. HE-1100 significantly increased (2.3 fold þ/-1.2 after 24h, p¼0.0444 and 2.3-fold þ/-1.0 after 72h, p¼0.0239) the CYR61 protein production by human OA chondrocytes. After 72h, HE-1100 slightly but not significantly increased aggrecan production by 14 ± 19 % (p¼0.1117) and significantly increased type II collagen pro-peptide production by 27 ± 20 % (p¼0.0147). For both time points CYR61 production by OA chondrocytes was positively and significantly correlated with aggrecan (r¼0.66, p¼0.0004) and type II collagen pro-peptide (r¼0.64, p¼0.0008) production. In alginate beads culture, pro-MMP-13 was significantly decreased by HE-1100 treated cultures from day 7 to day 14 (from-16 to-25 %, p<0.05) and from day 17 to 21 (-22 %, p¼0.0331) in comparison to controls. Conclusions: HE-1100 significantly modified the expression of DUSP1, C10orf10, ZFP36/L1 and CLEC3A, which are pathway mediators involved in MMP-13 expression and activation. Further, long-term (28 days) treatment with HE-1100 significantly reduced the production of pro-MMP-13, the inactive precursor of the metalloproteinase MMP-13 involved in type II collagen degradation. HE-1100 also promoted extracellular matrix formation probably through CYR61 production, a growth factor well correlated with type II collagen and aggrecan production.
Bone is one of the most common metastatic target sites in breast cancer, with more than 200 thous... more Bone is one of the most common metastatic target sites in breast cancer, with more than 200 thousand new cases of invasive cancer diagnosed in the US alone in 2011. We set out to establish a multimodality imaging platform for bone metastases in small animals as a tool to non-invasively quantify metastasis growth, imaging the ensuing bone lesions and possibly the response to treatment. To this end, a mouse model of osteolytic metastatic bone tumors was characterized with SPECT/CT and MRI over time. A cell line capable of forming bone metastases, MDA-MB-231, was genetically modified to stably express the reporter gene herpes simplex virus-1 thymidine kinase (hsv-1 tk). The intracellular accumulation of the radiolabeled tracer [ 123 I]FIAU promoted by HSV-1 TK specifically pinpoints the location of tumor cells which can be imaged in vivo by SPECT. First, a study using tumors implanted subcutaneously was performed. The SPECT/MRI overlays and the ex vivo γcounting showed a linear correlation in terms of %ID/cm 3 (R 2 = 0.93) and %ID/g (R 2 = 0.77), respectively. Then, bone metastasis growth was imaged weekly by SPECT/CT and T 2-weighted MRI over a maximum of 40 days postintracardiac injection of tumor cells. The first activity spots detectable with SPECT, around day 20 post-cell injection, were smaller than 2 mm 3 and not yet visible by MRI and increased in volume and in %ID over the weeks. Osteolytic bone lesions were visible by CT (in vivo) and μCT (ex vivo). The SPECT/MRI overlays also showed a linear correlation in terms of %ID/cm 3 (R 2 = 0.86). In conclusion, a new multimodality imaging platform has been established that non-invasively combines images of active tumor areas (SPECT), tumor volume (MRI) and the corresponding bone lesions (CT and μCT). To our knowledge this is the first report where the combination of soft tissue information from MRI, bone lesions by CT, and reporter gene imaging by SPECT is used to non-invasively follow metastatic bone lesions.
The use of contrast-enhanced MRI to enable in vivo specific characterization of atherosclerotic p... more The use of contrast-enhanced MRI to enable in vivo specific characterization of atherosclerotic plaques is increasing. In this study the intrinsic ability of two differently sized gadolinium-based contrast agents to enhance atherosclerotic plaques in ApoE(-/-) mice was evaluated with MRI. We obtained a kinetic profile for contrast enhancement, as the literature data on optimal imaging time points is scarce, and assessed the longer-term kinetics. Signal enhancement in the wall of the aortic arch, following intravenous injection of paramagnetic micelles and liposomes, was followed for 1 week. In vivo T(1)-weighted MRI plaque enhancement characteristics were complemented by fluorescence microscopy of NIR(664) incorporated in the contrast agents and quantification of tissue and blood Gd-DTPA. Both micelles and liposomes enhanced contrast in T(1)-weighted MR images of plaques in the aortic arch. The average contrast-to-noise ratio increased after liposome or micelle injection to 260 or 2...
This paper presents a fully automated method for atlas-based whole-body segmentation in non-contr... more This paper presents a fully automated method for atlas-based whole-body segmentation in non-contrastenhanced Micro-CT data of mice. The position and posture of mice in such studies may vary to a large extent, complicating data comparison in cross-sectional and follow-up studies. Moreover, Micro-CT typically yields only poor soft-tissue contrast for abdominal organs. To overcome these challenges, we propose a method that divides the problem into an atlas constrained registration based on high-contrast organs in Micro-CT (skeleton, lungs and skin), and a soft tissue approximation step for low-contrast organs. We first present a modification of the MOBY mouse atlas (Segars et al., 2004) by partitioning the skeleton into individual bones, by adding anatomically realistic joint types and by defining a hierarchical atlas tree description. The individual bones as well as the lungs of this adapted MOBY atlas are then registered one by one traversing the model tree hierarchy. To this end, we employ the Iterative Closest Point method and constrain the Degrees of Freedom of the local registration, dependent on the joint type and motion range. This atlas-based strategy renders the method highly robust to exceptionally large postural differences among scans and to moderate pathological bone deformations. The skin of the torso is registered by employing a novel method for matching distributions of geodesic distances locally, constrained by the registered skeleton. Because of the absence of image contrast between abdominal organs, they are interpolated from the atlas to the subject domain using Thin-Plate-Spline approximation, defined by correspondences on the already established registration of high-contrast structures (bones, lungs and skin). We extensively evaluate the proposed registration method, using 26 non-contrast-enhanced Micro-CT datasets of mice, and the skin registration and organ interpolation, using contrast-enhanced Micro-CT datasets of 15 mice. The posture and shape varied significantly among the animals and the data was acquired in vivo. After registration, the mean Euclidean distance was less than two voxel dimensions for the skeleton and the lungs respectively and less than one voxel dimension for the skin. Dice coefficients of volume overlap between manually segmented and interpolated skeleton and organs vary between 0.47 ± 0.08 for the kidneys and 0.73 ± 0.04 for the brain. These experiments demonstrate the method's effectiveness for overcoming exceptionally large variations in posture, yielding acceptable approximation accuracy even in the absence of soft-tissue contrast in in vivo Micro-CT data without requiring user initialization.
Breast cancer frequently metastasizes to bone, where tumor cells induce osteoclasts to locally de... more Breast cancer frequently metastasizes to bone, where tumor cells induce osteoclasts to locally destroy bone. During bone resorption, growth factors are locally released that may support bone metastatic growth. Differently from most other tissues, drugs that can limit local turnover, such as bisphosphonates and osteoprotegerin (OPG), are available for bone. We examined the hypothesis that inhibition of bone resorption by two different mechanisms may also affect the growth of cancer cells in bone. For this, we tested the effects of high doses of OPG and zoledronic acid (ZOL) on progression of MDA-231-B/Luc+ breast cancer cells in the bone microenvironment using whole body bioluminescent reporter imaging (BLI). Both treatments significantly inhibited the development of radiographically detectable osteolytic lesions. Histologic examination corroborated the radiographic findings, showing that both treatments preserved the integrity of bone trabeculae and prevented bone destruction (significantly higher trabecular bone volumes vs. vehicle). However, whereas practically no TRAcP-positive osteoclasts were observed in tibiae preparations of animals treated with Fc-OPG, TRAcP-positive osteoclasts were still present in the animals treated with ZOL. Intra-bone tumor burden was reduced with ZOL and Fc-OPG treatment. Although there appeared to be a trend for less overall total tumor burden upon treatment with both compounds, this was not significant as assessed by BLI and histomorphometric analysis due to the extramedullary growth of cancer cells which was not affected by these treatments. Collectively, anti-resorptive agents with different mechanisms of action-ZOL and OPGsignificantly reduced cancer-induced osteolysis and intra-osseous tumor burden, but failed to restrain local tumor growth. However, interference with the bone micro-environmental growth support could still be of therapeutic relevance when given to patients early in the course of bone metastatic disease.
Prolonged 17-estradiol (E2) administration affects insulin sensitivity. However, it is unknown wh... more Prolonged 17-estradiol (E2) administration affects insulin sensitivity. However, it is unknown whether E2 influences insulin sensitivity directly or indirectly e.g. via modulation of plasma free fatty acid levels and/or intra-hepatic lipid levels. Therefore, acute effects of E2 administration were studied by performing a hyperinsulinemic-euglycemic clamp in an insulin resistant mouse model (APOE*3-Leiden mice, which had been fed a high fat diet for 13 weeks). Six hours after E2 administration, estrogen receptor mediated transcription was induced predominantly in liver, but plasma triglyceride, insulin, free fatty acid and intrahepatic lipid levels were unaffected. During the hyperinsulinemic clamp, the hepatic glucose production was significantly inhibited in the E2 treated mice as compared to control mice (4.5 11 versus 34 29 mol·min·kg; P=0.013), whereas the peripheral glucose disposal rate was significantly lower in the E2 treated mice (29.8±8.9 versus 50.9±26.4 mol·min·kg ; P=0....
aligned to the human reference genome (UCSC hg19) to generate the transcriptome. Differential exp... more aligned to the human reference genome (UCSC hg19) to generate the transcriptome. Differential expression analysis between HE-1100 and placebo was made in R using the DESeq2 package to identify the differentially expressed genes in the OA-associated regulatory pathways. The protein production of the selected genes was quantified by ELISA in 10 independent human OA chondrocytes cultures. Results: According to the DESeq2 analysis, HE-1100 significantly modified the expression of 13 genes in OA chondrocytes by at least 10% with an adjusted p-value < 0.05 : EGR1 (þ93%), FOS (þ87%), NR4A1 (þ43%), DUSP1 (þ18%), ZFP36 (þ18%), ZFP36L1 (þ14%), NFKBIZ (þ16%) and CYR61 (þ14%) were upregulated and ATF7IP (-10%), TXNIP (-11%), C10orf10 (-12%), CLEC3A (-12%) and MMP13 (-18%) were downregulated after 24h HE-1100 treatment. HE-1100 significantly increased (2.3 fold þ/-1.2 after 24h, p¼0.0444 and 2.3-fold þ/-1.0 after 72h, p¼0.0239) the CYR61 protein production by human OA chondrocytes. After 72h, HE-1100 slightly but not significantly increased aggrecan production by 14 ± 19 % (p¼0.1117) and significantly increased type II collagen pro-peptide production by 27 ± 20 % (p¼0.0147). For both time points CYR61 production by OA chondrocytes was positively and significantly correlated with aggrecan (r¼0.66, p¼0.0004) and type II collagen pro-peptide (r¼0.64, p¼0.0008) production. In alginate beads culture, pro-MMP-13 was significantly decreased by HE-1100 treated cultures from day 7 to day 14 (from-16 to-25 %, p<0.05) and from day 17 to 21 (-22 %, p¼0.0331) in comparison to controls. Conclusions: HE-1100 significantly modified the expression of DUSP1, C10orf10, ZFP36/L1 and CLEC3A, which are pathway mediators involved in MMP-13 expression and activation. Further, long-term (28 days) treatment with HE-1100 significantly reduced the production of pro-MMP-13, the inactive precursor of the metalloproteinase MMP-13 involved in type II collagen degradation. HE-1100 also promoted extracellular matrix formation probably through CYR61 production, a growth factor well correlated with type II collagen and aggrecan production.
Bone is one of the most common metastatic target sites in breast cancer, with more than 200 thous... more Bone is one of the most common metastatic target sites in breast cancer, with more than 200 thousand new cases of invasive cancer diagnosed in the US alone in 2011. We set out to establish a multimodality imaging platform for bone metastases in small animals as a tool to non-invasively quantify metastasis growth, imaging the ensuing bone lesions and possibly the response to treatment. To this end, a mouse model of osteolytic metastatic bone tumors was characterized with SPECT/CT and MRI over time. A cell line capable of forming bone metastases, MDA-MB-231, was genetically modified to stably express the reporter gene herpes simplex virus-1 thymidine kinase (hsv-1 tk). The intracellular accumulation of the radiolabeled tracer [ 123 I]FIAU promoted by HSV-1 TK specifically pinpoints the location of tumor cells which can be imaged in vivo by SPECT. First, a study using tumors implanted subcutaneously was performed. The SPECT/MRI overlays and the ex vivo γcounting showed a linear correlation in terms of %ID/cm 3 (R 2 = 0.93) and %ID/g (R 2 = 0.77), respectively. Then, bone metastasis growth was imaged weekly by SPECT/CT and T 2-weighted MRI over a maximum of 40 days postintracardiac injection of tumor cells. The first activity spots detectable with SPECT, around day 20 post-cell injection, were smaller than 2 mm 3 and not yet visible by MRI and increased in volume and in %ID over the weeks. Osteolytic bone lesions were visible by CT (in vivo) and μCT (ex vivo). The SPECT/MRI overlays also showed a linear correlation in terms of %ID/cm 3 (R 2 = 0.86). In conclusion, a new multimodality imaging platform has been established that non-invasively combines images of active tumor areas (SPECT), tumor volume (MRI) and the corresponding bone lesions (CT and μCT). To our knowledge this is the first report where the combination of soft tissue information from MRI, bone lesions by CT, and reporter gene imaging by SPECT is used to non-invasively follow metastatic bone lesions.
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Papers by Clemens Lowik