**5.2 Tumor vasculature and ultrastructure**

258 Advances in the Biology, Imaging and Therapies for Glioblastoma

Fig. 2. MR images used to calculate tumor volumes and tumor growth. T2-weighted images

of a mouse GL261 glioma at 14 (A), 23 (B) and 26 days (C) following intracerebral implantation of cells. Tumors are outlined (white ellipses). (D) Calculated GL261 tumor

Fig. 3. Determination of necrotic volumes from MR image-observed tumors. (A) T2 weighted MR image of a GL261 mouse glioma at 23 days following intracerebral

lesions. (B) Necrotic volumes from tumors can be measured (n=5; meanS.D.). (C)

Corresponding histological slide depicting necrosis in a GL261 tumor.

implantation of cells. Note dark void regions in the heterogeneous tumor which are necrotic

Tumor morphology can also provide information on tumor invasiveness and necrotic lesions. Necrotic lesions are depicted as dark void regions in a tumor (as shown in Fig. 3A), of which volumes (e.g. Fig. 3B) can be measured from multiple slices (Towner *et al*., 2010a). A comparison between the orthotopic rat glioma models, C6 and RG2, and the chemical

volumes which follow an exponential increase over time.

MR angiography can provide information on new blood vessels formed in tumors, a process known as angiogenesis which is required to maintain tumor growth. On small animal imaging MRI systems, the image in-plane resolution is >50 µm, which allows visualization of major blood vessels, arterioles and venules (Doblas *et al*., 2008, 2010). Quantitation of brain and tumor blood vessels can also be obtained, as well as measurements on blood vessel diameters and lengths (Doblas *et al*., 2008, 2010). An increase in total brain tumor blood volume was found to directly correlate with increasing tumor volumes during tumor growth (Doblas *et al*., 2010).

Quantification of the Brownian motion of water or diffusion within tissues can be measured through the apparent diffusion coefficient (ADC) which is obtained from diffusion-weighted imaging datasets. DWI yields ultrastructural information on cellular density and the extracellular matrix (Waldman *et al*., 2009; Sadeghi *et al*., 2003). Figure 5 shows an example of an ADC (Fig. 5 b) map in a C6 glioma-bearing rat brain, indicating higher ADC values in tumor tissue compared to contralateral 'normal' brain tissue (Garteiser *et al*., 2010).

Temporal diffusion spectroscopy based on oscillating gradient spin-echo (OGSE) MRI was used to detect microscopic structural variations at the subcellular scale in C6 rat gliomas

Fig. 4. MR angiography of a GL261 mouse glioma. (A) T2-weighted MR image of a GL261 glioma-bearing mouse brain (23 days following intracerebral implantation of cells.) (B) and (C), 3 dimensional angiograms of mouse brain blood vessels of a GL261 glioma-bearing mouse. Note altered vasculature on left-hand side in image B, as well as increased blood vessels (middle cerebral artery; depicted in left-mid-region of image C) in the tumor region.

Assessment of Rodent Glioma Models Using Magnetic Resonance Imaging Techniques 261

smaller relative increase in vessel size index in the RG2 tumor (Valable *et al*., 2008). The vessel density eventually decreased with increasing tumor cell proliferation (Valable *et al*., 2008). It was thought that early expression of Ang-2, MMP-2 and MMP-9, which has been found in C6 gliomas, could account for the destabilization of vessel walls and a reduction in

Magnetic resonance spectroscopy (MRS) is an MR method that allows regional metabolite levels to be measured in tumors compared to surrounding non-tumor tissue. Important metabolites that can be assessed by 1H-MRS in brain tumors include N-acetyl aspartate (NAA), total creatine (tCr), total choline (tCho), lactate, *myo*-inositol and mobile lipids associated with necrosis. Metabolite levels can be quantified as metabolite ratios or absolute concentrations, or analyzed using pattern recognition (Waldman *et al*., 2009). Figure 6 depicts an example of regional MR spectra obtained in a rat C6 glioma within tumor and contralateral 'normal' brain tissues. Glioma tissue has characteristic increased mobile lipid signals at 1.3 and 0.9 ppm, as well as decreased NAA, tCr and tCho, compared to surrounding 'normal' brain tissue. Metabolites measured by MRS can provide information on brain tissue status, such as (1) NAA being a marker of healthy neuronal integrity, which is compromised within brain tumors, (2) choline-containing compounds being associated with cell membrane turnover, (3) creatinecontaining compounds being associated with the cellular energy status, (4) lactate being linked to anaerobic respiration, and (5) mobile lipids resulting from intracellular lipid droplets and necrosis (Waldman *et al*., 2009). Spatially resolved 1H-MRS was used to reveal significantly decreased levels of NAA and tCr and increased lactate (or lipids) in intracerebral rat C6 gliomas, compared to the contralateral hemisphere (Ross *et al*., 1992; Terpstra *et al*., 1996). In F98 rat gliomas there were detected increases in tCho, *myo*-inositol and lipids, as well as the

Fig. 6. MR spectroscopy of a rat C6 glioma. Regional (PRESS; point-resolved spectroscopy; 333 mm3 or 27 µl volume) was obtained in tumor (right region in T2-weighted; bottom spectrum) and 'normal' (left region; top spectrum) brain tissues (19 days post-intracerebral implantation of C6 cells). Peak assignments: (1) tCho (total choline), (2) tCr (total creatine), (3) NAA (N-acetyl aspartate), (4) methylene (-CH2-)n lipid hydrogens, and (5) methyl (-CH3)

vessel density during C6 tumor growth (Valable *et al*., 2008).

absence of a NAA signal (Gyngell *et al*., 1994).

lipid hydrogens.

**5.3 Tumor metabolism** 

Fig. 5. Diffusion- and perfusion-weighted imaging of a rat C6 glioma. (A) T2-weighted MR image of a C6 glioma-bearing rat brain (20 days following intracerebral implantation of cells). (B) ADC map (110-4 mm2/s) of a C6 glioma. Note higher ADC values in the tumor compared to 'normal' brain tissue. (C) Perfusion map (ml/(100gmin)) in a C6 glioma. Note decreased perfusion values in tumor tissue, compared to 'normal' brain tissue.

(Colvin *et al*., 2008). An extension of DWI is diffusion tensor imaging (DTI) which obtains multidirectional images that can be used to obtain diffusional directionality information quantified as fractional anisotropy and displayed as dominant white fiber tract maps (tractography) ((Waldman *et al*., 2009). DTI was used to differentiate between two rat glioma models, which observed C6 glioma-induced ischemia of tumor-surrounding tissues, compared to the more infiltrative nature of F98 gliomas that penetrated into the corpus callosum (Asanuma *et al*., 2008a, 2008b).

Arterial spin labeling (ASL), a perfusion-weighted MRI method, uses a MR image signal based on the influx of magnetically labeled water into blood, as a means of quantifying absolute levels of cerebral blood flow (CBF) (Waldman *et al*., 2009). Another widely used perfusion method is dynamic susceptibility contrast MRI (DSC-MRI), which can be used to measure relative cerebral blood volume, relative cerebral blood flow (rCBF) and mean transit time from the kinetics resulting from a change in signal intensity following the bolus administration of a gadolinium (Gd)-based contrast agent (Waldman *et al*., 2009). Steadystate susceptibility contrast (SSC) images were used to obtain MRI vessel caliber index (VCI) measurements in a xenograft orthotopic U87 mouse brain tumor model, and were found to correlate closely with intravital optical microscopy (IVM) measurements (Farrar *et al*., 2010). Dynamic contrast-enhanced MRI (DCE-MRI) is commonly used to measure the permeability of the blood-brain-barrier (BBB). The transfer coefficient, Ktrans is associated with endothelial permeability, vascular surface area and blood flow ((Waldman *et al*., 2009). DCE-MRI revealed a significant change in tumor vessel permeability that was dependent on tumor progression and size in a GL26 orthotopic mouse glioblastoma model (Veeravagu *et al*., 2008). Fig. 5C depicts decreased perfusion values in a C6 glioma, compared to contralateral 'normal' brain tissue, obtained using the ASL method. Perfusion values were found to be increased in the more aggressive RG2 rat glioma compared to C6 (Towner *et al*., 2010a), which is associated with increased vascular proliferation in this model. The increased perfusion in the RG2 model was also correlated with increased capillary vascularity visualized in 3D confocal microscopy fluorescence images, as well as more diffuse and smaller blood vessels observed by MRA, compared to the C6 model (Towner *et al*., 2010a). A study by Valable *et al*. demonstrated that there was a slight reduction in vessel density in the tumor center within RG2 gliomas compared to a more increased reduction in vessel density within C6 gliomas, which was characterized by an increased blood volume fraction, and a smaller relative increase in vessel size index in the RG2 tumor (Valable *et al*., 2008). The vessel density eventually decreased with increasing tumor cell proliferation (Valable *et al*., 2008). It was thought that early expression of Ang-2, MMP-2 and MMP-9, which has been found in C6 gliomas, could account for the destabilization of vessel walls and a reduction in vessel density during C6 tumor growth (Valable *et al*., 2008).

#### **5.3 Tumor metabolism**

260 Advances in the Biology, Imaging and Therapies for Glioblastoma

Fig. 5. Diffusion- and perfusion-weighted imaging of a rat C6 glioma. (A) T2-weighted MR image of a C6 glioma-bearing rat brain (20 days following intracerebral implantation of cells). (B) ADC map (110-4 mm2/s) of a C6 glioma. Note higher ADC values in the tumor compared to 'normal' brain tissue. (C) Perfusion map (ml/(100gmin)) in a C6 glioma. Note

(Colvin *et al*., 2008). An extension of DWI is diffusion tensor imaging (DTI) which obtains multidirectional images that can be used to obtain diffusional directionality information quantified as fractional anisotropy and displayed as dominant white fiber tract maps (tractography) ((Waldman *et al*., 2009). DTI was used to differentiate between two rat glioma models, which observed C6 glioma-induced ischemia of tumor-surrounding tissues, compared to the more infiltrative nature of F98 gliomas that penetrated into the corpus

Arterial spin labeling (ASL), a perfusion-weighted MRI method, uses a MR image signal based on the influx of magnetically labeled water into blood, as a means of quantifying absolute levels of cerebral blood flow (CBF) (Waldman *et al*., 2009). Another widely used perfusion method is dynamic susceptibility contrast MRI (DSC-MRI), which can be used to measure relative cerebral blood volume, relative cerebral blood flow (rCBF) and mean transit time from the kinetics resulting from a change in signal intensity following the bolus administration of a gadolinium (Gd)-based contrast agent (Waldman *et al*., 2009). Steadystate susceptibility contrast (SSC) images were used to obtain MRI vessel caliber index (VCI) measurements in a xenograft orthotopic U87 mouse brain tumor model, and were found to correlate closely with intravital optical microscopy (IVM) measurements (Farrar *et al*., 2010). Dynamic contrast-enhanced MRI (DCE-MRI) is commonly used to measure the permeability of the blood-brain-barrier (BBB). The transfer coefficient, Ktrans is associated with endothelial permeability, vascular surface area and blood flow ((Waldman *et al*., 2009). DCE-MRI revealed a significant change in tumor vessel permeability that was dependent on tumor progression and size in a GL26 orthotopic mouse glioblastoma model (Veeravagu *et al*., 2008). Fig. 5C depicts decreased perfusion values in a C6 glioma, compared to contralateral 'normal' brain tissue, obtained using the ASL method. Perfusion values were found to be increased in the more aggressive RG2 rat glioma compared to C6 (Towner *et al*., 2010a), which is associated with increased vascular proliferation in this model. The increased perfusion in the RG2 model was also correlated with increased capillary vascularity visualized in 3D confocal microscopy fluorescence images, as well as more diffuse and smaller blood vessels observed by MRA, compared to the C6 model (Towner *et al*., 2010a). A study by Valable *et al*. demonstrated that there was a slight reduction in vessel density in the tumor center within RG2 gliomas compared to a more increased reduction in vessel density within C6 gliomas, which was characterized by an increased blood volume fraction, and a

decreased perfusion values in tumor tissue, compared to 'normal' brain tissue.

callosum (Asanuma *et al*., 2008a, 2008b).

Magnetic resonance spectroscopy (MRS) is an MR method that allows regional metabolite levels to be measured in tumors compared to surrounding non-tumor tissue. Important metabolites that can be assessed by 1H-MRS in brain tumors include N-acetyl aspartate (NAA), total creatine (tCr), total choline (tCho), lactate, *myo*-inositol and mobile lipids associated with necrosis. Metabolite levels can be quantified as metabolite ratios or absolute concentrations, or analyzed using pattern recognition (Waldman *et al*., 2009). Figure 6 depicts an example of regional MR spectra obtained in a rat C6 glioma within tumor and contralateral 'normal' brain tissues. Glioma tissue has characteristic increased mobile lipid signals at 1.3 and 0.9 ppm, as well as decreased NAA, tCr and tCho, compared to surrounding 'normal' brain tissue. Metabolites measured by MRS can provide information on brain tissue status, such as (1) NAA being a marker of healthy neuronal integrity, which is compromised within brain tumors, (2) choline-containing compounds being associated with cell membrane turnover, (3) creatinecontaining compounds being associated with the cellular energy status, (4) lactate being linked to anaerobic respiration, and (5) mobile lipids resulting from intracellular lipid droplets and necrosis (Waldman *et al*., 2009). Spatially resolved 1H-MRS was used to reveal significantly decreased levels of NAA and tCr and increased lactate (or lipids) in intracerebral rat C6 gliomas, compared to the contralateral hemisphere (Ross *et al*., 1992; Terpstra *et al*., 1996). In F98 rat gliomas there were detected increases in tCho, *myo*-inositol and lipids, as well as the absence of a NAA signal (Gyngell *et al*., 1994).

Fig. 6. MR spectroscopy of a rat C6 glioma. Regional (PRESS; point-resolved spectroscopy; 333 mm3 or 27 µl volume) was obtained in tumor (right region in T2-weighted; bottom spectrum) and 'normal' (left region; top spectrum) brain tissues (19 days post-intracerebral implantation of C6 cells). Peak assignments: (1) tCho (total choline), (2) tCr (total creatine), (3) NAA (N-acetyl aspartate), (4) methylene (-CH2-)n lipid hydrogens, and (5) methyl (-CH3) lipid hydrogens.

Assessment of Rodent Glioma Models Using Magnetic Resonance Imaging Techniques 263

indicative of the invasive nature of a tumor. The distribution of c-Met was found to be more widely dispersed, but mainly concentrated in peri-tumor regions (Towner *et al*., 2008, 2010c). Figure 7A depicts the contrast-enhancement in a C6 glioma 3 hours following i.v. administration of a Gd-DTPA-albumin-anti-c-Met-biotin probe, and the corresponding perfusion map showing the increased uptake of the anti-c-Met probe in the peri-tumor

iNOS levels were found to vary in different rat glioma models, as detected with a Gd-DTPA-albumin-anti-iNOS-biotin (anti-iNOS) probe, where percent MRI signal intensity changes were highest in the C6 tumor, compared to the RG2 and ENU-induced tumors (Towner *et al*., 2010a). Dynamic kinetic monitoring of the anti-iNOS probe indicated sustained uptake over 3 hours within tumor tissue regions, and no specific uptake of a control Gd-DTPA-albumin-IgG-biotin contrast agent within tumors (Towner *et al*., 2010a). Fluorescence imaging of the anti-iNOS probe by targeting the biotin moiety with streptavidin-Cy3, verified higher levels of probe uptake in C6 tumors versus RG2 gliomas, despite the increased perfusion and micro-vascularity detected in the RG2 tumors (Towner *et al*., 2010a). Confirmation of the presence of iNOS in glioma cell membrane, but not in normal astrocytes, was obtained by transmission electron microscopy of gold-labeled anti-

Fig. 7. Molecular MR imaging of c-Met levels in a rat C6 glioma. (A) T1-weighted MR image 3 hours following i.v. administration of a Gd-DTPA-albumin-anti-c-Met-biotin probe. Note contrast enhancement in peri-tumor regions. (B) Perfusion map depicting distribution of the anti-c-Met probe. (C) Illustration of the Gd-DTPA-albumin-anti-c-Met-biotin probe, with

Clinically, therapeutic response to surgical resection of gliomas, followed by radiation and chemotherapy, can be assessed by dynamic contrast-enhanced morphological MRI, increases in ADC values detected by DWI (Waerzeggers *et al*., 2010), decreases in the fractional tumor volume with a corresponding low relative cerebral blood volume detected by perfusion imaging, and/or reduced choline levels detected by MRS (Waldman *et al*., 2009). DCE-MRI was used to establish reduced Gd enhancement consistent with decreased vascular permeability following i.v. bevacizumab and carboplatin therapy in a human glioma (UW28) nude rat model (Jahnke *et al*., 2009). DCE-MRI using a high molecular weight contrast agent, albumin-Gd-DTPA, showed significantly increased Ktrans at the rim of

regions (Towner *et al*., 2008).

iNOS antibodies (Towner *et al*., 2010a).

the antibody (Ab) conjugated to albumin.

**6. MRI evaluation of therapeutics against gliomas** 

Other atomic nuclei, other than 1H, have also been used to assess 13C and 19F containing compounds in rodent gliomas. Hyperpolarized 13C MR metabolic imaging was used to follow the metabolism of hyperpolarized [1-(13)C]-pyruvate to lactate in rats with human glioblastoma xenografts (U-251 MG and U-87 MG), indicating higher levels in tumor versus normal brain tissue, and variations between tumor models (Park *et al*., 2010). Rat 9L glioma cells labeled with perfluoro-15-crown-5-ether *ex vivo* and implanted into rat striatum was used to measure intracellular partial pressure of oxygen (pO2) (oximetry) in tumors (Kadayakkara *et al*., 2010).

#### **5.4 Molecular imaging**

The concept used in molecular imaging is to couple a targeting moiety (antibody or peptide targeted to a protein of interest) to a reporter molecule, such as a MRI contrast agent. Two commonly used MRI contrast agents are gadolinium (Gd)-based compounds, or iron oxidebased nanoparticles. The targeted MR probes are often injected via a tail-vein in rats or mice. The expression of cell adhesion molecules, such as integrins, has been found to be upregulated during tumor growth and angiogenesis, and αVβ3 expression which has been correlated with tumor aggressiveness, can be measured by MRI with targeted paramagneticlabeled cyclic arginine-glycine-aspartic acid (RGD) peptides (Sipkins *et al*., 1998; Waerzeggers *et al*., 2010). Within U87MG xenograft tumors in nude mice, RGD-labeled ultrasmall superparamagnetic iron oxide (USPIO) probes were found to accumulate only within the neovasculature associated with tumors, but not within tumor cells (Kiessling *et al*., 2009). Tumor angiogenesis was also monitored via the expression of CD105 in F98 tumor-bearing rats with the use of Gd-DTPA liposomes targeted to CD105 (CD105-Gd-SLs) and MR imaging (Zhang *et al*., 2009). Combined MRI-coupled fluorescence tomography was used to assess epidermal growth factor receptor (EGFR) status in high- and low-EGFR expression tumor cells injected into nude mice by measuring the levels of a near-infrared fluorophore bound to a EGF ligand (Davis *et al*., 2010).

MR imaging probes have also been developed to monitor *in vivo* levels of other angiogenic proteins known to be over-expressed in malignant brain tumors, such as VEGF-R2 (vascular endothelial growth factor receptor 2) (He *et al*., 2010; Towner *et al*., 2010b); a tumor cell migration/invasion marker, such as c-Met, a tyrosine kinase receptor for the scatter factor (also known as the hepatocyte growth factor) (Towner *et al*., 2008, 2010c); and the inflammatory marker, inducible nitric oxide synthase (iNOS) (Towner *et al*., 2010a). With the use of a Gd-DTPA-albumin-anti-VEGFR2-biotin probe, regional differences in VEGFR2 levels were detected by MRI *in vivo* in a C6 glioma model, and probe-specificity for glioma tissue, particularly in the peri-tumor and peri-necrotic regions, was confirmed by tagging the biotin moiety of the probe in excised tissues with streptavidin-Cy3 (He *et al*., 2010). The control non-specific probe had rat IgG conjugated to the albumin, instead of the VEGFR2 antibody. A similar result was obtained when an aminated dextran-coated iron-oxide nanoparticles conjugated with a VEGFR2 antibody was used in a C6 glioma model, where distribution of the probe was mainly in the peri-tumor and peri-necrotic regions of the tumor (He *et al*., 2010). Confirmation of the presence of the nanoprobes was obtained by using Prussian blue stain for the VEGFR2-targeting iron oxide nanoparticles in excised tumor tissues (He *et al*., 2010).

Both Gd- and iron oxide-based probes were also developed to characterize c-Met levels in C6 gliomas. c-Met is a tumor marker that is over-expressed in many malignant cancers,

Other atomic nuclei, other than 1H, have also been used to assess 13C and 19F containing compounds in rodent gliomas. Hyperpolarized 13C MR metabolic imaging was used to follow the metabolism of hyperpolarized [1-(13)C]-pyruvate to lactate in rats with human glioblastoma xenografts (U-251 MG and U-87 MG), indicating higher levels in tumor versus normal brain tissue, and variations between tumor models (Park *et al*., 2010). Rat 9L glioma cells labeled with perfluoro-15-crown-5-ether *ex vivo* and implanted into rat striatum was used to measure intracellular partial pressure of oxygen (pO2) (oximetry) in tumors

The concept used in molecular imaging is to couple a targeting moiety (antibody or peptide targeted to a protein of interest) to a reporter molecule, such as a MRI contrast agent. Two commonly used MRI contrast agents are gadolinium (Gd)-based compounds, or iron oxidebased nanoparticles. The targeted MR probes are often injected via a tail-vein in rats or mice. The expression of cell adhesion molecules, such as integrins, has been found to be upregulated during tumor growth and angiogenesis, and αVβ3 expression which has been correlated with tumor aggressiveness, can be measured by MRI with targeted paramagneticlabeled cyclic arginine-glycine-aspartic acid (RGD) peptides (Sipkins *et al*., 1998; Waerzeggers *et al*., 2010). Within U87MG xenograft tumors in nude mice, RGD-labeled ultrasmall superparamagnetic iron oxide (USPIO) probes were found to accumulate only within the neovasculature associated with tumors, but not within tumor cells (Kiessling *et al*., 2009). Tumor angiogenesis was also monitored via the expression of CD105 in F98 tumor-bearing rats with the use of Gd-DTPA liposomes targeted to CD105 (CD105-Gd-SLs) and MR imaging (Zhang *et al*., 2009). Combined MRI-coupled fluorescence tomography was used to assess epidermal growth factor receptor (EGFR) status in high- and low-EGFR expression tumor cells injected into nude mice by measuring the levels of a near-infrared

MR imaging probes have also been developed to monitor *in vivo* levels of other angiogenic proteins known to be over-expressed in malignant brain tumors, such as VEGF-R2 (vascular endothelial growth factor receptor 2) (He *et al*., 2010; Towner *et al*., 2010b); a tumor cell migration/invasion marker, such as c-Met, a tyrosine kinase receptor for the scatter factor (also known as the hepatocyte growth factor) (Towner *et al*., 2008, 2010c); and the inflammatory marker, inducible nitric oxide synthase (iNOS) (Towner *et al*., 2010a). With the use of a Gd-DTPA-albumin-anti-VEGFR2-biotin probe, regional differences in VEGFR2 levels were detected by MRI *in vivo* in a C6 glioma model, and probe-specificity for glioma tissue, particularly in the peri-tumor and peri-necrotic regions, was confirmed by tagging the biotin moiety of the probe in excised tissues with streptavidin-Cy3 (He *et al*., 2010). The control non-specific probe had rat IgG conjugated to the albumin, instead of the VEGFR2 antibody. A similar result was obtained when an aminated dextran-coated iron-oxide nanoparticles conjugated with a VEGFR2 antibody was used in a C6 glioma model, where distribution of the probe was mainly in the peri-tumor and peri-necrotic regions of the tumor (He *et al*., 2010). Confirmation of the presence of the nanoprobes was obtained by using Prussian blue stain for the VEGFR2-targeting iron oxide nanoparticles in excised

Both Gd- and iron oxide-based probes were also developed to characterize c-Met levels in C6 gliomas. c-Met is a tumor marker that is over-expressed in many malignant cancers,

(Kadayakkara *et al*., 2010).

**5.4 Molecular imaging** 

fluorophore bound to a EGF ligand (Davis *et al*., 2010).

tumor tissues (He *et al*., 2010).

indicative of the invasive nature of a tumor. The distribution of c-Met was found to be more widely dispersed, but mainly concentrated in peri-tumor regions (Towner *et al*., 2008, 2010c). Figure 7A depicts the contrast-enhancement in a C6 glioma 3 hours following i.v. administration of a Gd-DTPA-albumin-anti-c-Met-biotin probe, and the corresponding perfusion map showing the increased uptake of the anti-c-Met probe in the peri-tumor regions (Towner *et al*., 2008).

iNOS levels were found to vary in different rat glioma models, as detected with a Gd-DTPA-albumin-anti-iNOS-biotin (anti-iNOS) probe, where percent MRI signal intensity changes were highest in the C6 tumor, compared to the RG2 and ENU-induced tumors (Towner *et al*., 2010a). Dynamic kinetic monitoring of the anti-iNOS probe indicated sustained uptake over 3 hours within tumor tissue regions, and no specific uptake of a control Gd-DTPA-albumin-IgG-biotin contrast agent within tumors (Towner *et al*., 2010a). Fluorescence imaging of the anti-iNOS probe by targeting the biotin moiety with streptavidin-Cy3, verified higher levels of probe uptake in C6 tumors versus RG2 gliomas, despite the increased perfusion and micro-vascularity detected in the RG2 tumors (Towner *et al*., 2010a). Confirmation of the presence of iNOS in glioma cell membrane, but not in normal astrocytes, was obtained by transmission electron microscopy of gold-labeled antiiNOS antibodies (Towner *et al*., 2010a).

Fig. 7. Molecular MR imaging of c-Met levels in a rat C6 glioma. (A) T1-weighted MR image 3 hours following i.v. administration of a Gd-DTPA-albumin-anti-c-Met-biotin probe. Note contrast enhancement in peri-tumor regions. (B) Perfusion map depicting distribution of the anti-c-Met probe. (C) Illustration of the Gd-DTPA-albumin-anti-c-Met-biotin probe, with the antibody (Ab) conjugated to albumin.
