High-contrast Tumor Research Articles

Targeting and imaging of HER2 overexpression tumor with a new peptide-based 68Ga-PET radiotracer

Human epidermal growth factor receptor 2 (HER2) overexpression, as a predictive biomarker, is associated with more tumor aggressiveness and worse clinical outcomes in cancer, whereas it’s accurate identification has led to the choice of effective treatments in many patients. In this study, a peptide-based PET probe (68Ga-DOTA-(Ser)3-LTVSPWY) was developed for imaging HER2 expression in tumors. The DOTA-(Ser)3-LTVSPWY was labeled with 68Ga and then was evaluated in vitro with HER2-positive SKOV-3 cell line; moreover, the in vivo biodistribution and PET/CT imaging were performed in xenografted tumor-bearing nude mice. The 68Ga-DOTA-(Ser)3-LTVSPWY displayed the high radiochemical purity greater than 95% and good stability in normal saline and human serum. The cellular binding experiments showed that the cell uptake in HER2-positive ovarian cancer cells could be effectively blocked by non-labeled peptide. The Kd and Bmax values for radiolabeled peptide were obtained at 2.5 ± 0.6 nM and (3.4 ± 0.2) × 105 sites per cell, respectively. Biodistribution study demonstrated that tumor-to-blood and tumor-to-muscle ratios were about 1.73 ± 0.36 and 3.78 ± 0.17 at 120 min after the injection of the radiolabeled peptide, respectively. Tumor imaging by PET/CT exhibited high contrast tumor image at 60 min after injection in animal models. Consequently, the results were indicative of the specific accumulation of 68Ga-DOTA-(Ser)3-LTVSPWY peptide in HER2-positive tumors and the suitability of its application as a PET probe for the diagnosis of HER2-overexpression tumor.

One pot synthesis of zwitteronic 99mTc doped ultrasmall iron oxide nanoparticles for SPECT/T1-weighted MR dual-modality tumor imaging

In this study, we have synthesized 99mTc intrinsically labeled ultrasmall magnetic iron oxide nanoparticles with zwitterionic surface coating (99mTc-ZW-USIONPs) via one pot synthesis using sulfobetains functionalized poly (acrylic acid) as stabilizer and Na99mTcO4 and SnCl2 as additives. The commercialization of single photon emission computed tomography (SPECT)/magnetic resonance imaging (MRI) scanner made the combination use of 99mTc and iron oxide nanoparticles attracting much attention. Direct doping radioisotope into nanoparticles has the advantages of excellent radiochemical stability and no restriction on the surface functionalization. The complex Technetium chemistry made it challenging to direct dope 99mTc into IONPs, especially those ultrasmall ones without precipitation. We proved that it is possible to prepare 99mTc doped USIONPs with excellent water solubility and favorable T1 signal by controlling the radioactivity and reducing agent amount. With no need of chelator, the zwitterionic surface resists the protein corona formation, resulting in a reduced RES uptake and higher tumor contrast. The 99mTc-ZW-USIONPs demonstrated excellent performance of tumor SPECT and T1-weighted MR imaging capability in 4T1 tumor bearing mice. Together with their ease of preparation and superior biocompatibility, we believe these 99mTc-ZW-USIONPs represent a type of promising dual contrast agent for SPECT/T1 MRI.

A preclinical PET dual-tracer imaging protocol for ER and HER2 phenotyping in breast cancer xenografts

BackgroundNuclear medicine is on the constant search of precision radiopharmaceutical approaches to improve patient management. Although discordant expression of the estrogen receptor (ER) and the human epidermal growth factor receptor 2 (HER2) in breast cancer is a known dilemma for appropriate patient management, traditional tumor sampling is often difficult or impractical. While 2-deoxy-2[18F]fluoro-D-glucose (18F-FDG)-positron emission tomography (PET) is an option to detect subclinical metastases, it does not provide phenotype information. Radiolabeled antibodies are able to specifically target expressed cell surface receptors. However, their long circulating half-lives (days) require labeling with long-lived isotopes, such as 89Zr, in order to allow sufficient time for tracer clearance from the blood compartment and to accumulate adequately in target tumors and, thus, generate high-quality PET images. The aim of this study was to develop a dual-tracer PET imaging approach consisting of a fast-clearing small molecule and a slow-clearing antibody. This approach was evaluated in a model consisting of mice harboring separate breast cancer xenografts with either an ER+/HER2− or ER−/HER2+ phenotype, comparable to human metastatic disease with intertumor heterogeneity. Lastly, the aim of our study was to determine the feasibility of specifically identifying these two important phenotypes in an acceptable time window.MethodsFemale nude mice were subcutaneously implanted on opposite shoulders with the ER+/HER2− and ER−/HER2+ MCF-7 and JIMT-1 tumor cell lines, respectively. A second model was developed consisting of mice implanted orthotopically with either MCF-7 or JIMT-1 cells. Pharmacokinetic analysis, serial PET imaging, and biodistribution were first performed for [89Zr]Zr-DFO-trastuzumab (89Zr-T) up to 8 days post-injection (p.i.) in JIMT-1 bearing mice. Region-of-interest (ROI) and biodistribution-derived uptake (% injected-activity/gram of tissue [%IA/g]) values and tumor-to-background ratios were obtained. Results were compared in order to validate ROI and identify early time points that provided high contrast tumor images. For the dual-tracer approach, cohorts of tumor-bearing mice were then subjected to sequential tracer PET imaging. On day 1, mice were administered 4-fluoro-11β-methoxy-16α-[18F]-fluoroestradiol (4FMFES) which targets ER and imaged 45 min p.i. This was immediately followed by the injection of 89Zr-T. Mice were then imaged on day 3 or day 7. ROI analysis was performed, and uptake was calculated in tumors and selected healthy organs for all radiotracers. Quality of tumor targeting for all tracers was evaluated by tumor contrast visualization, tumor and normal tissue uptake, and tumor-to-background ratios.Results89Zr-T provided sufficiently high tumor and low background uptake values that furnished high contrast tumor images by 48 h p.i. For the dual-tracer approach, 4FMFES provided tumor uptake values that were significantly increased in MCF-7 tumors. When 89Zr-T-PET was combined with 18F-4FMFES-PET, the entire dual-tracer sequential-imaging procedure provided specific high-quality contrast images of ER+/HER2− MCF-7 and ER−/HER2+ JIMT-1 tumors for 4FMFES and 89Zr-T, respectively, as short as 72 h from start to finish.ConclusionsThis protocol can provide high contrast images of tumors expressing ER or HER2 within 3 days from injection of 4FMFES to final scan of 89Zr-T and, hence, provides a basis for future dual-tracer combinations that include antibodies.

A super-resolution framework for the reconstruction of T2-weighted (T2w) time-resolved (TR) 4DMRI using T1w TR-4DMRI as the guidance.

The purpose of this study was to develop T2-weighted (T2w) time-resolved (TR) four-dimensional magnetic resonance imaging (4DMRI) reconstruction technique with higher soft-tissue contrast for multiple breathing cycle motion assessment by building a super-resolution (SR) framework using the T1w TR-4DMRI reconstruction as guidance. The multi-breath T1w TR-4DMRI was reconstructed by deforming a high-resolution (HR: 2×2×2mm3 ) volumetric breath-hold (BH, 20s) three-dimensional magnetic resonance imaging (3DMRI) image to a series of low-resolution (LR:5×5×5mm3 ) 3D cine images at a 2Hz frame rate in free-breathing (FB, 40s) using an enhanced Demons algorithm, namely [T1BH →FB] reconstruction. Within the same imaging session, respiratory-correlated (RC) T2w 4DMRI (2×2×2mm3 ) was acquired based on an internal navigator to gain HR T2w (T2HR ) in three states (full exhalation and mid and full inhalation) in ~5min. Minor binning artifacts in the RC-4DMRI were automatically identified based on voxel intensity correlation (VIC) between consecutive slices as outliers (VIC<VICmean -σ) and corrected by deforming the artifact slices to interpolated slices from the adjacent slices iteratively until no outliers were identified. A T2HR image with minimal deformation (<1cm at the diaphragm) from the T1BH image was selected for multi-modal B-Spline deformable image registration (DIR) to establish the T2HR -T1BH voxel correspondence. Two approaches to reconstruct T2w TR-4DMRI were investigated: (A) T2HR →[T1BH →FB]: to deform T2w HR to T1w BH only as T1w TR-4DMRI was reconstructed, and combine the two displacement vector fields (DVFs) to reconstruct T2w TR-4DMRI, and (B) [T2HR ←T1BH ]→FB: to deform T1w BH to T2w HR first and apply the deformed T1w BH to reconstruct T2w TR-4DMRI. The reconstruction times were similar, 8-12min per volume. To validate the two methods, T2w- and T1w-mapped 4D XCAT digital phantoms were utilized with three synthetic spherical tumors (ϕ=2.0, 3.0, and 4.0cm) in the lower or mid lobes as the ground truth to evaluate the tumor location (the center of mass, COM), size (volume ratio, %V), and shape (Dice index). Six lung cancer patients were scanned under an IRB-approved protocol and the T2w TR-4DMRI images reconstructed from the two methods were compared based on the preservation of the three tumor characteristics. The local tumor-contained image quality wasalso characterized using the VIC and structure similarity (SSIM)indexes. In the 4D digital phantom, excellent tumor alignment after T2HR -T1HR DIR is achieved: ∆COM=0.8±0.5mm, %V=1.06±0.02, and Dice=0.91±0.03, in both deformation directions using the DIR-target image as the reference. In patients, binning artifacts are corrected with improved image quality: average VIC increases from 0.92±0.03 to 0.95±0.01. Both T2w TR-4DMRI reconstruction methods produce similar tumor alignment errors ∆COM=2.9±0.6mm. However, method B ([T2HR ←T1BH ]→FB) produces superior results in preserving more T2w tumor features with a higher %V=0.99±0.03, Dice=0.81±0.06, VIC=0.85±0.06, and SSIM=0.65±0.10 in the T2w TR-4DMRI images. This study has demonstrated the feasibility of T2w TR-4DMRI reconstruction with high soft-tissue contrast and adequately-preserved tumor position, size, and shape in multiple breathing cycles. The T2w-centric DIR (method B) produces a superior solution for the SR-based framework of T2w TR-4DMRI reconstruction with highly preserved tumor characteristicsand local image features, which are useful for tumor delineation and motion management in radiation therapy.

68Ga-PSMA-11 PET/CT in patients with recurrent prostate cancer-a modified protocol compared with the common protocol.

68Ga-PSMA-11 PET/CT is commonly performed at 1 h post injection (p.i.). However, various publications have demonstrated that most prostate cancer (PC) lesions exhibit higher contrast at later imaging. The aim of this study was to compare the "common" protocol of 68Ga-PSMA-11 PET/CT with a modified protocol. In 2017, we used the following scanning protocol for 68Ga-PSMA-11 PET/CT in patients with recurrent PC: acquisition at 1 h p.i. without further preparations. From 2018, all scans were conducted at 1.5 h p.i. In addition, patients were orally hydrated with 1 L of water 0.5 h p.i. and were injected with 20 mg of furosemide 1 h p.i. Both protocols including 112 patients (2017) and 156 (modified protocol in 2018) were retrospectively compared. Rates of pathologic scans, maximum standardized uptake values (SUVmax), and tumor contrast (ratio lesion-SUVmax/background-SUVmean) as well as average standardized uptake values (SUVmean) of urinary bladder were analyzed. Both tumor contrast and tracer uptake were significantly (p < 0.001) higher in the novel protocol. Although statistically not significant, the rates of pathologic scans were also higher in the modified protocol: 76.3% vs. 68.8% for all PSA values including 38.9% vs. 25.0% for PSA < 0.5 ng/ml and 60.0% vs. 56.7% for PSA > 0.5-≤ 2.0 ng/ml. Average SUVmean of the urinary bladder was significantly (p < 0.001) lower with the modified protocol. The modified protocol, which includes a combination of delayed image acquisition at 1.5 h p.i., hydration, and furosemide resulted in higher tumor contrast and seems to have the potential to increase the rates of pathological scans, especially at low PSA levels.

NIR-II-Excited Intravital Two-Photon Microscopy Distinguishes Deep Cerebral and Tumor Vasculatures with an Ultrabright NIR-I AIE Luminogen.

Intravital fluorescence imaging of vasculature morphology and dynamics in the brain and in tumors with large penetration depth and high signal-to-background ratio (SBR) is highly desirable for the study and theranostics of vascular-related diseases and cancers. Herein, a highly bright fluorophore (BTPETQ) with long-wavelength absorption and aggregation-induced near-infrared (NIR) emission (maximum at ≈700 nm) is designed for intravital two-photon fluorescence (2PF) imaging of a mouse brain and tumor vasculatures under NIR-II light (1200 nm) excitation. BTPETQ dots fabricated via nanoprecipitation show uniform size of around 42 nm and a high quantum yield of 19 ± 1% in aqueous media. The 2PF imaging of the mouse brain vasculatures labeled by BTPETQ dots reveals a 3D blood vessel network with an ultradeep depth of 924 µm. In addition, BTPETQ dots show enhanced 2PF in tumor vasculatures due to their unique leaky structures, which facilitates the differentiation of normal blood vessels from tumor vessels with high SBR in deep tumor tissues. Moreover, the extravasation and accumulation of BTPETQ dots in deep tumor (more than 900 µm) is visualized under NIR-II excitation. This study highlights the importance of developing NIR-II light excitable efficient NIR fluorophores for in vivo deep tissue and high contrast tumor imaging.

Albumin-chaperoned cyanine dye yields superbright NIR-II fluorophore with enhanced pharmacokinetics.

NIR-II fluorescence imaging greatly reduces scattering coefficients for nearly all tissue types at long wavelengths, benefiting deep tissue imaging. However, most of the NIR-II fluorophores suffer from low quantum yields and/or short circulation time that limit the quality of NIR-II imaging. Here, we engineered a supramolecular assembly of protein complex with lodged cyanine dyes to produce a brilliant NIR-II fluorophore, providing a NIR-II quantum yield of 21.2% with prolonged circulation time. Computational modeling revealed the mechanism for fluorescence enhancement and identified key parameters governing albumin complex for NIR-II fluorophores. Our complex afforded high-resolution microvessel imaging, with a 3-hour imaging window compared to 2 min for free dye alone. Furthermore, the complexation strategy was applied to an antibody-derived assembly, offering high-contrast tumor imaging without affecting the targeting ability of the antibody. This study provides a facile strategy for producing high-performance NIR-II fluorophores by chaperoning cyanine dyes with functional proteins.

Clinical Results in Medullary Thyroid Carcinoma Suggest High Potential of Pretargeted Immuno-PET for Tumor Imaging and Theranostic Approaches.

Monoclonal antibody (mAb)-based therapies have experienced considerable growth in cancer management. When labeled with radionuclides, mAbs also represent promising probes for imaging or theranostic approaches. Initially, mAbs have been radiolabeled with single-photon emitters, such as 131I, 99mTc, or 111In, for diagnostic purposes or to improve radioimmunotherapy (RIT) using dosimetry estimations. Today, more accurate imaging is achieved using positron- emission tomography (PET). Thanks to the important technical advances in the production of PET emitters and their related radiolabeling methods, the last decade has witnessed the development of a broad range of new probes for specific PET imaging. Immuno-PET, which combines the high sensitivity and resolution of a PET camera with the specificity of a monoclonal antibody, is fully in line with this approach. As RIT, immuno-PET can be performed using directly radiolabeled mAbs or using pretargeting to improve imaging contrast. Pretargeted immuno-PET has been developed against different antigens, and promising results have been reported in tumor expressing carcinoembryonic antigen (CEA; CEACAM5) using a bispecific mAb (BsmAb) and a radiolabeled peptide. Medullary thyroid carcinoma (MTC) is an uncommon thyroid cancer subtype which accounts for <10% of all thyroid neoplasms. Characterized by an intense expression of CEA, MTC represents a relevant tumor model for immuno-PET. High sensitivity of pretargeted immunoscintigraphy using murine or chimeric anti-CEA BsMAb and pretargeted haptens-peptides labeled with 111In or 131I were reported in metastatic MTC patients 20 years ago. Recently, an innovative clinical study reported high tumor uptake and contrast using pretargeted anti-CEA immuno-PET in relapsed MTC patients. This review focuses on MTC as an example, but the same pretargeting technique has been applied with success for clinical PET imaging of other CEA-expressing tumors and other pretargeting systems. In particular, those exploiting bioorthogonal chemistry also appear interesting in preclinical animal models, suggesting the high potential of pretargeting for diagnostic and theranostic applications.

One-Step 18F-Labeling and Preclinical Evaluation of Prostate-Specific Membrane Antigen Trifluoroborate Probes for Cancer Imaging.

After the identification of the high-affinity glutamate-ureido scaffold, the design of several potent 18F- and 68Ga-labeled tracers has allowed spectacular progress in imaging recurrent prostate cancer by targeting the prostate-specific membrane antigen (PSMA). We evaluated a series of PSMA-targeting probes that are 18F-labeled in a single step for PET imaging of prostate cancer. Methods: We prepared 8 trifluoroborate constructs for prostate cancer imaging, to study the influence of the linker and the trifluoroborate prosthetic on pharmacokinetics and image quality. After 1-step labeling by 19F–18F isotopic exchange, the radiotracers were injected in mice bearing LNCaP xenografts, with or without blocking controls, to assess specific uptake. PET/CT images and biodistribution data were acquired at 1 h after injection and compared with 18F-DCFPyL on the same mouse strain and tumor model. Results: All tracers exhibited nanomolar affinities, were labeled in good radiochemical yields at high molar activities, and exhibited high tumor uptake in LNCaP xenografts with clearance from nontarget organs. Most derivatives with a naphthylalanine linker showed significant gastrointestinal excretion. A radiotracer incorporating this linker with a dual trifluoroborate-glutamate labeling moiety showed high tumor uptake, low background activity, and no liver or gastrointestinal track accumulation. Conclusion: PSMA-targeting probes with trifluoroborate prosthetic groups represent promising candidates for prostate cancer imaging because of facile labeling while affording high tumor uptake values and contrast ratios that are similar to those obtained with 18F-DCFPyL.

Mn-Loaded apolactoferrin dots forin vivoMRI and NIR-II cancer imaging

The dual modal imaging probeH-dotwas prepared forin vivoNIR-II/MR imaging of orthotopic U87MG glioblastoma xenografts.

Small, Long Blood Half-Life Iodine Nanoparticle for Vascular and Tumor Imaging

Standard clinical X-ray contrast agents are small iodine-containing molecules that are rapidly cleared by the kidneys and provide robust imaging for only a few seconds, thereby limiting more extensive vascular and tissue biodistribution imaging as well as optimal tumor uptake. They are also not generally useful for preclinical microCT imaging where longer scan times are required for high resolution image acquisition. We here describe a new iodine nanoparticle contrast agent that has a unique combination of properties: 20 nm hydrodynamic diameter, covalent PEG coating, 40 hour blood half-life, 50% liver clearance after six months, accumulation in tumors, and well-tolerated to at least 4 g iodine/kg body weight after intravenous administration in mice. These characteristics are unique among the other iodine nanoparticles that have been previously reported and provide extended-time high contrast vascular imaging and tumor loading. As such, it is useful for preclinical MicroCT animal studies. Potential human applications might include X-ray radiation dose enhancement for cancer therapy and vascular imaging for life-threatening situations where high levels of contrast are needed for extended periods of time.

Bispecific affibody molecule targeting HPV16 and HPV18E7 oncoproteins for enhanced molecular imaging of cervical cancer.

High-risk human papillomavirus (HPV16 and HPV18) are now widely recognized as responsible for cervical cancer, which remains to be the most common gynecologic malignancy in women worldwide. It is well known that viral oncoproteins E6/E7 play key roles in HPV-associated cervical carcinogenesis. Thus, in vivo detection of the two oncoproteins may provide important diagnostic information influencing patient management. More recently, affibody molecules have been demonstrated to be a promising candidate for development as molecular imaging probes. Based on the two monomeric affibody molecules (ZHPV16E7 and ZHPV18E7) generated in our laboratory, here, we used a peptide linker (Gly4Ser)3 to link ZHPV16E7 and ZHPV18E7 to develop a novel heterodimeric affibody ZHPV16E7-(Gly4Ser)3-ZHPV18E7. Both biosensor and immunofluorescence assays have proved that the heterodimeric affibody molecule targeted simultaneously HPV16 and HPV18E7 proteins by binding to the viral oncoproteins. In vivo tumor-imaging experiments using the Dylight755-labeled heterodimeric affibody revealed that strongly high-contrast tumor retention of the heterodimers occurred in both HPV16- and HPV18-derived tumors of nude mice 0.5h post-injection. The accumulation of Dylight755-labeled heterodimers in tumors was achieved over 48h. Therefore, we believe that this novel heterodimeric affibody molecule has great potential utility in molecular imaging in vivo and diagnosis of HPV-associated cervical cancers.

Proteomic Analysis and NIR-II Imaging of MCM2 Protein in Hepatocellular Carcinoma.

Targeted therapy of hepatocellular carcinoma (HCC) is essential for improved therapies. Therefore, identification of key targets specifically to HCC is an urgent requirement. Herein, an iTRAQ quantitative proteomic approach was employed to identify differentially expressed proteins in HCC tumor tissues. Of the upregulated tumor-related proteins, minichromosome maintenance 2 (MCM2), a DNA replication licensing factor, was one of the most significantly altered proteins, and its overexpression was confirmed using tissue microarray. Clinicopathological analysis of multiple cohorts of HCC patients indicated that overexpression of MCM2 was validated in 89.8% tumor tissues and strongly correlated with clinical stage. Furthermore, siRNA-mediated repression of MCM2 expression resulted in significant suppression of the HepG2 cell cycle and proliferation through the cyclin D-dependent kinases (CDKs) 2/7 pathway. Finally, the first small molecule-based MCM2-targeted NIR-II probe CH1055-MCM2 was concisely generated and subsequently evaluated in mice bearing HepG2 xenografts. The excellent imaging properties such as good tumor uptake and high tumor contrast and specificity were achieved in the small animal models. This analytical strategy can determine novel accessible targets of HCC useful for imaging and therapy.

Hypoxia-triggered single molecule probe for high-contrast NIR II/PA tumor imaging and robust photothermal therapy.

Hypoxia is a common characteristic of solid tumors. This important feature is associated with resistance to radio-chemotherapy, which results in poor prognosis and probability of tumor recurrence. Taking advantage of background-free NIR II fluorescence imaging and deeper-penetrating photoacoustic (PA) imaging, we developed a hypoxia-triggered and nitroreductase (NTR) enzyme-responsive single molecule probe for high-contrast NIR II/PA tumor imaging and hypoxia-activated photothermal therapy (PTT), which will overcome cellular resistance during hypoxia.Methods: The single molecule probe IR1048-MZ was synthesized by conjugating a nitro imidazole group as a specific hypoxia trigger with an IR-1048 dye as a NIR II/PA signal reporter. We investigated the NIR II fluorescence, NIR absorbance and photothermal effect in different hypoxia conditions in vitro, and performed NIR II/PA tumor imaging and hypoxia-activated photothermal therapy in mice.Results: This versatile molecular probe IR1048-MZ not only realized high-contrast tumor visualization with a clear boundary by NIR II fluorescence imaging, but also afforded deep-tissue penetration at the centimeter level by 3D PA imaging. Moreover, after being activated by NTR that is overexpressed in hypoxic tumors, the probe exhibited a significant photothermal effect for curative tumor ablation with no recurrence.Conclusions: We have developed the first hypoxia-triggered and NTR enzyme-responsive single molecule probe for high-contrast NIR II/PA tumor imaging and hypoxia-activated photothermal therapy. By tracing the activity of NTR, IR1048-MZ may be a promising contrast agent and theranostic formulation for other hypoxia-related diseases (such as cancer, inflammation, stroke, and cardiac ischemia).

An improved 99mTc-HYNIC-(Ser)3-LTVSPWY peptide with EDDA/tricine as co-ligands for targeting and imaging of HER2 overexpression tumor

Overexpression of human epidermal receptor 2 (HER2) has given the opportunity for targeting and delivering of imaging radiotracers. The aim of this study was to evaluate the 99mTc-HYNIC-(EDDA/tricine)-(Ser)3-LTVSPWY peptide for tumor targeting and imaging of tumor with overexpression of HER2. The HYNIC-(Ser)3-LTVSPWY was labeled with 99mTc in presence of EDDA/tricine mixture as co-ligands. The in vitro and in vivo studies of this radiolabeled peptide were performed for cellular specific binding and tumor targeting. The high radiochemical purity of 99mTc-HYNIC (EDDA/tricine)-(Ser)3-LTVSPWY was obtained to be 99%. It exhibited high stability in normal saline and human serum. In HER2 binding affinity study, a significant reduction in uptake of radiolabeled peptide (7.7 fold) was observed by blocking SKOV-3 cells receptors with unlabeled peptide. The KD and Bmax values for this radiolabeled peptide were determined as 3.3 ± 1.0 nM and 2.9 ± 0.3 × 106 CPM/pMol, respectively. Biodistribution study revealed tumor to blood and tumor to muscle ratios about 6.9 and 4 respectively after 4 h. Tumor imaging by gamma camera demonstrated considerable high contrast tumor uptake. This developed 99mTc-HYNIC-(Ser)3-LTVSPWY peptide selectively targeted on HER2 tumor and exhibited a high target uptake combined with acceptable low background activity for tumor imaging in mice. The results of this study and its comparison with another study showed that 99mTc-HYNIC-(EDDA/tricine)-(Ser)3-LTVSPWY is much better than previously reported radiolabeled peptide as 99mTc-CSSS-LTVSPWY for HER2 overexpression tumor targeting and imaging.

Indocyanine Green-Labeled Polysarcosine for in Vivo Photoacoustic Tumor Imaging

Photoacoustic (PA) imaging has been considered an attractive imaging modality for sensitive and in-depth imaging of biomolecules with a high resolution in vivo. PA imaging probes utilizing fluorescence dyes, including indocyanine green (ICG), have been proposed to enhance PA signal intensity. On the other hand, nanomicelles modified with polysarcosine (PSar), a biocompatible hydrophilic polymer, on their surface have previously achieved rapid tumor uptake, suggesting active transport of PSar into tumor tissues. Thus, we hypothesized that PSar-based materials might be utilized as diagnostic probes for targeting tumors and therefore evaluated the potential of PSar labeled with an ICG derivative, ICG-PSar, as a PA imaging probe for targeting cancer. In this study, ICG-PSars with differing molecular weights (10, 20, and 30 kDa) were synthesized. In vitro cellular uptake studies using ICG-PSar demonstrated rapid uptake in colon26 tumor cells partially via macropinocytosis-mediated endocytosis. In vivo fluorescence imaging and biodistribution study indicated that ICG-PSar30k exhibited high accumulation in the tumor (8.4% dose/g), with high tumor-to-blood ratios reaching 4.6 at 24 h post injection of the probe. Finally, in vivo PA imaging studies showed that PA signal increased in tumors (251%) but not in blood vessels, achieving high contrast tumor imaging at 24 h after ICG-PSar30k probe injection. These results suggest that ICG-PSar has potential as a tumor-targeting PA imaging probe.

Dynamic Volume Assessment of Hepatocellular Carcinoma in Rat Livers Using a Clinical 3T MRI and Novel Segmentation

ABSTRACTPurpose: In vivo liver cancer research commonly uses rodent models. One of the limitations of such models is the lack of accurate and reproducible endpoints for a dynamic assessment of growing tumor nodules. The aim of this study was to validate a noninvasive, true volume segmentation method using two rat hepatocellular carcinoma (HCC) models, correlating magnetic resonance imaging (MRI) with histological volume measurement, and with blood levels of α-fetoprotein. Materials and methods: We used 3T clinical MRI to quantify tumor volume with follow-up over time. Using two distinct rat HCC models, calculated MRI tumor volumes were correlated with volumes from histological sections, or with blood levels of α-fetoprotein. Eleven rats, comprising six Buffalo rats (n = 9 scans) and five Fischer rats (n = 14 tumors), were injected in the portal vein with 2.5 × 105 and 2.0 × 106 syngeneic HCC cells, respectively. Longitudinal (T1) relaxation time- and transverse (T2) relaxation time-weighted MR images were acquired. Results: The three-dimensional (3D) T1-weighted gradient echo had 0.35-mm isotropic resolution allowing accurate semi-automatic volume segmentation. 2D T2-weighted imaging provided high tumor contrast. Segmentation of combined 3D gradient echo T1-weighted images and 2D turbo spin echo T2-weighted images provided excellent correlation with histology (y = 0.866x + 0.034, R² = 0.997 p < .0001) and with α-fetoprotein (y = 0.736x + 1.077, R² = 0.976, p < .0001). There was robust inter- and intra-observer reproducibility (intra-class correlation coefficient > 0.998, p < .0001). Conclusions: We have developed a novel, noninvasive contrast imaging protocol which enables semi-automatic 3D volume quantification to analyze nonspherical tumor nodules and to follow up the growth of tumor nodules over time.

Generation of affibody molecules specific for HPV16 E7 recognition.

Cervical cancer caused by infection with high-risk human papillomavirus remains to be the most deadly gynecologic malignancy worldwide. It is well documented that persistent expression of two oncogenes (E6/E7) plays the key roles in cervical cancer. Thus, in vivo detection of the oncoproteins is very important for the diagnosis of the cancer. Recently, affibody molecules have been demonstrated to be a powerful targeting probe for tumor–targeted imaging and diagnosis. In this study, four HPV16 E7-binding affibody molecules (ZHPV16 E7127, ZHPV16E7301, ZHPV16E7384 and ZHPV16E7745) were screened from a phage-displayed peptide library and used for molecular imaging in tumor-bearing mice. Biosensor binding analyses showed first that the four affibody molecules bound to HPV16 E7 with very high affinity and specificity. They co-localized with E7 protein only in two HPV16-positive cancer cells (SiHa and CaSki). Furthermore, affibody ZHPV16E7384 was conjugated with Dylight755 and used for in vivo tumor-imaging. Strongly high-contrast tumor retention of this affibody only occurred in HPV16-derived tumors of mice as early as 30 min post-injection, not in HPV-negative and HPV18-derived tumors. The accumulation of Dylight755-conjugated ZHPV16E7384 in tumor was achieved over a longer time period (24 h). The data here provide strong evidence that E7-specific affibody molecules have great potential used for molecular imaging and diagnosis of HPV-induced cancers.

Multi-Modal Imaging in a Mouse Model of Orthotopic Lung Cancer.

BackgroundInvestigation of CF800, a novel PEGylated nano-liposomal imaging agent containing indocyanine green (ICG) and iohexol, for real-time near infrared (NIR) fluorescence and computed tomography (CT) image-guided surgery in an orthotopic lung cancer model in nude mice.MethodsCF800 was intravenously administered into 13 mice bearing the H460 orthotopic human lung cancer. At 48 h post-injection (peak imaging agent accumulation time point), ex vivo NIR and CT imaging was performed. A clinical NIR imaging system (SPY®, Novadaq) was used to measure fluorescence intensity of tumor and lung. Tumor-to-background-ratios (TBR) were calculated in inflated and deflated states. The mean Hounsfield unit (HU) of lung tumor was quantified using the CT data set and a semi-automated threshold-based method. Histological evaluation using H&E, the macrophage marker F4/80 and the endothelial cell marker CD31, was performed, and compared to the liposomal fluorescence signal obtained from adjacent tissue sectionsResultsThe fluorescence TBR measured when the lung is in the inflated state (2.0 ± 0.58) was significantly greater than in the deflated state (1.42 ± 0.380 (n = 7, p<0.003). Mean fluorescent signal in tumor was highly variable across samples, (49.0 ± 18.8 AU). CT image analysis revealed greater contrast enhancement in lung tumors (a mean increase of 110 ± 57 HU) when CF800 is administered compared to the no contrast enhanced tumors (p = 0.0002).ConclusionPreliminary data suggests that the high fluorescence TBR and CT tumor contrast enhancement provided by CF800 may have clinical utility in localization of lung cancer during CT and NIR image-guided surgery.

18F]Fluoro-azomycin-2´-deoxy-β-d-ribofuranoside — A new imaging agent for tumor hypoxia in comparison with [18F]FAZA

IntroductionRadiolabeled 2-nitroimidazoles (azomycins) are a prominent class of biomarkers for PET imaging of hypoxia. [18F]Fluoro-azomycin-α-arabinoside ([18F]FAZA) – already in clinical use – may be seen as α-configuration nucleoside, but enters cells only via diffusion and is not transported by cellular nucleoside transporters. To enhance image contrast in comparison to [18F]FAZA our objective was to 18F-radiolabel an azomycin-2´-deoxyriboside with β-configuration ([18F]FAZDR, [18F]-β-8) to mimic nucleosides more closely and comparatively evaluate it versus [18F]FAZA. MethodsPrecursor and cold standards for [18F]FAZDR were synthesized from methyl 2-deoxy-d-ribofuranosides α- and β-1 in 6 steps yielding precursors α- and β-5. β-5 was radiolabeled in a GE TRACERlab FXF-N synthesizer in DMSO and deprotected with NH4OH to give [18F]FAZDR ([18F]-β-8). [18F]FAZA or [18F]FAZDR was injected in BALB/c mice bearing CT26 colon carcinoma xenografts, PET scans (10min) were performed after 1, 2 and 3h post injection (p.i.). On a subset of mice injected with [18F]FAZDR, we analyzed biodistribution. Results[18F]FAZDR was obtained in non-corrected yields of 10.9±2.4% (9.1±2.2GBq, n=4) 60min EOB, with radiochemical purity >98% and specific activity >50GBq/μmol. Small animal PET imaging showed a decrease in uptake over time for both [18F]FAZDR (1h p.i.: 0.56±0.22% ID/cc, 3h: 0.17±0.08% ID/cc, n=9) and [18F]FAZA (1h: 1.95±0.59% ID/cc, 3h: 0.87±0.55% ID/cc), whereas T/M ratios were significantly higher for [18F]FAZDR at 1h (2.76) compared to [18F]FAZA (1.69, P<0.001), 3h p.i. ratios showed no significant difference. Moreover, [18F]FAZDR showed an inverse correlation between tracer uptake in carcinomas and oxygen breathing, while muscle tissue uptake was not affected by switching from air to oxygen. ConclusionsFirst PET imaging results with [18F]FAZDR showed advantages over [18F]FAZA regarding higher tumor contrast at earlier time points p.i. Availability of precursor and cold fluoro standard together with high output radiosynthesis will allow for a more detailed quantitative evaluation of [18F]FAZDR, especially with regard to mechanistic studies whether active transport processes are involved, compared to passive diffusion as observed for [18F]FAZA.