ImmunoPET imaging of TIGIT in the glioma microenvironment.

Glioblastoma (GBM) is the most common primary malignant brain tumor. Currently, there are few effective treatment options for GBM beyond surgery and chemo-radiation, and even with these interventions, median patient survival remains poor. While immune checkpoint inhibitors (ICIs) have demonstrated therapeutic efficacy against non-central nervous system cancers, ICI trials for GBM have typically had poor outcomes. TIGIT is an immune checkpoint receptor that is expressed on activated T-cells and has a role in the suppression of T-cell and Natural Killer (NK) cell function. As TIGIT expression is reported as both prognostic and a biomarker for anti-TIGIT therapy, we constructed a molecular imaging agent, [89Zr]Zr-DFO-anti-TIGIT (89Zr-αTIGIT), to visualize TIGIT in preclinical GBM by immunoPET imaging. PET imaging and biodistribution analysis of 89Zr-αTIGIT demonstrated uptake in the tumor microenvironment of GBM-bearing mice. Blocking antibody and irrelevant antibody tracer studies demonstrated specificity of 89Zr-αTIGIT with significance at a late time point post-tracer injection. However, the magnitude of 89Zr-αTIGIT uptake in tumor, relative to the IgG tracer was minimal. These findings highlight the features and limitations of using 89Zr-αTIGIT to visualize TIGIT in the GBM microenvironment.

Survival following complete resection of neuroblastoma in novel orthotopic rat xenograft model.

Neuroblastoma accounts for 15% of pediatric cancer deaths, despite multimodal therapy including surgical resection. Current neuroblastoma rodent models are insufficient for studying the impact of surgery and combination treatments, largely due to the small size of mouse models. Human neuroblastoma SK-N-BE(2) cells were injected into the left adrenal gland of 5-6-week-old RNU homozygous nude rats. Rats were either monitored by MRI until humane endpoint was reached or after 5 weeks underwent operative tumor resection, followed by monitoring for recurrence and survival. Following neuroblastoma cell implantation, the majority of tumors grew to greater than 5000 mm3 within 5.5-6.5 weeks, meeting the humane endpoint. Surgical resection was successfully done in 8 out of 9 rats, extending survival following tumor implantation from a median of 42 days to 78 days (p < 0.005). Pathology was consistent with human neuroblastoma, showing small round blue cell tumors with Homer-Wright rosettes, high mitoses and karyorrhectic index, and strong PHOX2B staining. Thus, we have established a novel orthotopic xenograft rat model of neuroblastoma and demonstrated increased survival of rats after surgical tumor resection. This model can be used for the development of surgical techniques, such as the use of intraoperative molecular imaging or assessment of combination therapies that include surgery.

Novel theranostic agent for PET imaging and targeted radiopharmaceutical therapy of tumour-infiltrating immune cells in glioma.

BACKGROUND: Malignant gliomas are deadly tumours with few therapeutic options. Although immunotherapy may be a promising therapeutic strategy for treating gliomas, a significant barrier is the CD11b+ tumour-associated myeloid cells (TAMCs), a heterogeneous glioma infiltrate comprising up to 40% of a glioma's cellular mass that inhibits anti-tumour T-cell function and promotes tumour progression. A theranostic approach uses a single molecule for targeted radiopharmaceutical therapy (TRT) and diagnostic imaging; however, there are few reports of theranostics targeting the tumour microenvironment. METHODS: Utilizing a newly developed bifunctional chelator, Lumi804, an anti-CD11b antibody (αCD11b) was readily labelled with either Zr-89 or Lu-177, yielding functional radiolabelled conjugates for PET, SPECT, and TRT. FINDINGS: 89Zr/177Lu-labeled Lumi804-αCD11b enabled non-invasive imaging of TAMCs in murine gliomas. Additionally, 177Lu-Lumi804-αCD11b treatment reduced TAMC populations in the spleen and tumour and improved the efficacy of checkpoint immunotherapy. INTERPRETATION: 89Zr- and 177Lu-labeled Lumi804-αCD11b may be a promising theranostic pair for monitoring and reducing TAMCs in gliomas to improve immunotherapy responses. FUNDING: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Molecular Imaging of Very Late Antigen-4 in Acute Lung Injury.

Inflammation plays a central role in the pathogenesis of acute lung injury (ALI) during both the acute pneumonitis stage and progression into the chronic fibroproliferative phase, leading to pulmonary fibrosis. Currently, there is an unmet clinical and research need for noninvasive ways to monitor lung inflammation through targeting of immunoregulatory pathways contributing to ALI pathogenesis. In this study, we evaluated the role of targeted imaging of very late antigen-4 (VLA-4), as a key integrin mediating the adhesion and recruitment of immune cells to inflamed tissues, in quantifying lung inflammation in a mouse model of lipopolysaccharide-induced ALI. Methods: ALI was induced by a single intratracheal administration of lipopolysaccharide (10, 20, or 40 µg per mouse) in C57BL/6J mice. Control mice were intratracheally instilled with sterile phosphate-buffered saline. VLA-4-targeted PET/CT was performed 24 h after intravenous injection of a 64Cu-labeled high-affinity peptidomimetic ligand referred to as 64Cu-LLP2A, which is conjugated with the chelator (1,4,8,11-tetraazacyclotetradecane-1-(methane phosphonic acid)-8-(methane carboxylic acid) and a polyethylene glycol 4 linker, at day 2 after the induction of ALI. Ex vivo biodistribution of 64Cu-LLP2A was determined by γ-counting of harvested organs. The severity of lung inflammation was assessed histologically and by measuring the expression of inflammatory markers in the lung tissue lysates using reverse transcription quantitative polymerase chain reaction. Results: Intratracheal lipopolysaccharide instillation led to an acute inflammatory response in the lungs, characterized by increased expression of multiple inflammatory markers and infiltration of myeloid cells, along with a significant and specific increase in 64Cu-LLP2A uptake, predominantly in a peribronchial distribution. There was a strong correlation between the lipopolysaccharide dose and 64Cu-LLP2A uptake, as quantified by in vivo PET (R = 0.69, P < 0.01). Expression levels of both subunits of VLA-4, that is, integrins α4 and ß1, significantly correlated with the expression of multiple inflammatory markers, including tumor necrosis factor-α, interleukin-1ß, and nitric oxide synthase-2, highlighting the potential of VLA-4 as a surrogate marker of acute lung inflammation. Notably, in vivo 64Cu-LLP2A uptake significantly correlated with the expression of multiple inflammatory markers and VLA-4. Conclusion: Our study demonstrates the feasibility of molecular imaging of VLA-4, as a mechanistically relevant target in ALI, and the accuracy of VLA-4-targeted PET in quantification of ongoing lung inflammation in a murine model.

Preclinical ImmunoPET Imaging of Glioblastoma-Infiltrating Myeloid Cells Using Zirconium-89 Labeled Anti-CD11b Antibody.

PURPOSE: Glioblastoma is a lethal brain tumor, heavily infiltrated by tumor-associated myeloid cells (TAMCs). TAMCs are emerging as a promising therapeutic target as they suppress anti-tumor immune responses and promote tumor cell growth. Quantifying TAMCs using non-invasive immunoPET could facilitate patient stratification for TAMC-targeted treatments and monitoring of treatment efficacy. As TAMCs uniformly express the cell surface marker, integrin CD11b, we evaluated a Zr-89 labeled anti-CD11b antibody for non-invasive imaging of TAMCs in a syngeneic orthotopic mouse glioma model. PROCEDURES: A human/mouse cross-reactive anti-CD11b antibody (clone M1/70) was conjugated to a DFO chelator and radiolabeled with Zr-89. PET/CT and biodistribution with or without a blocking dose of anti-CD11b Ab were performed 72 h post-injection (p.i.) of [89Zr]anti-CD11b Ab in mice bearing established orthotopic syngeneic GL261 gliomas and in non tumor-bearing mice. Flow cytometry and immunohistochemistry of dissected GL261 tumors were conducted to confirm the presence of CD11b+ TAMCs. RESULTS: Significant uptake of [89Zr]anti-CD11b Ab was detected at the tumor site (SUVmean = 2.60 ± 0.24) compared with the contralateral hemisphere (SUVmean = 0.6 ± 0.11). Blocking with a 10-fold lower specific activity of [89Zr]anti-CD11b Ab markedly reduced the SUV in the right brain (SUVmean = 0.11 ± 0.06), demonstrating specificity. Spleen and lymph nodes (myeloid cell rich organs) also showed high uptake of the tracer, and biodistribution analysis correlated with the imaging results. CD11b expression within the tumor was validated using flow cytometry and immunohistochemistry, which showed high CD11b expression primarily in the tumoral hemisphere compared with the contralateral hemisphere with very minimal accumulation in non tumor-bearing brain. CONCLUSION: These data establish that [89Zr]anti-CD11b Ab immunoPET targets CD11b+ cells (TAMCs) with high specificity in a mouse model of GBM, demonstrating the potential for non-invasive quantification of tumor-infiltrating CD11b+ immune cells during disease progression and immunotherapy in patients with GBM.

RAGE-specific single chain Fv for PET imaging of pancreatic cancer.

Noninvasive detection of both early pancreatic neoplasia and metastases could enhance strategies to improve patient survival in this disease that is notorious for an extremely poor prognosis. There are almost no identifiable targets for non-invasive diagnosis by positron emission tomography (PET) for patients with pancreatic ductal adenocarcinoma (PDAC). Over-expression of the receptor for advanced glycation end products (RAGE) is found on the cell surface of both pre-neoplastic lesions and invasive PDAC. Here, a RAGE-specific single chain (scFv) was developed, specific for PET imaging in syngeneic mouse models of PDAC. An anti-RAGE scFv conjugated with a sulfo-Cy5 fluorescence molecule showed high affinity and selectivity for RAGE expressing pancreatic tumor cells and genetically engineered KRASG12D mouse models of PDAC. An in vivo biodistribution study was performed with the 64Cu-radiolabled scFv in a syngeneic murine pancreatic cancer model, demonstrating both the feasibility and potential of an anti-RAGE scFv for detection of PDAC. These studies hold great promise for translation into the clinic.

Imaging the Tumor Microenvironment.

The tumor microenvironment consists of tumor, stromal, and immune cells, as well as extracellular milieu. Changes in numbers of these cell types and their environments have an impact on cancer growth and metastasis. Non-invasive imaging of aspects of the tumor microenvironment can provide important information on the aggressiveness of the cancer, whether or not it is metastatic, and can also help to determine early response to treatment. This chapter provides an overview on non-invasive in vivo imaging in humans and mouse models of various cell types and physiological parameters that are unique to the tumor microenvironment. Current clinical imaging and research investigation are in the areas of nuclear imaging (positron emission tomography (PET) and single photon emission computed tomography (SPECT)), magnetic resonance imaging (MRI) and optical (near infrared (NIR) fluorescence) imaging. Aspects of the tumor microenvironment that have been imaged by PET, MRI and/or optical imaging are tumor associated inflammation (primarily macrophages and T cells), hypoxia, pH changes, as well as enzymes and integrins that are highly prevalent in tumors, stroma and immune cells. Many imaging agents and strategies are currently available for cancer patients; however, the investigation of novel avenues for targeting aspects of the tumor microenvironment in pre-clinical models of cancer provides the cancer researcher with a means to monitor changes and evaluate novel treatments that can be translated into the clinic.

Preclinical immunoPET/CT imaging using Zr-89-labeled anti-PD-L1 monoclonal antibody for assessing radiation-induced PD-L1 upregulation in head and neck cancer and melanoma.

Radiation therapy (RT) can induce upregulation of programmed death ligand 1 (PD-L1) on tumor cells or myeloid cells, which may affect response to PD-1-based immunotherapy. PD-L1 upregulation during RT is a dynamic process that has been difficult to monitor during treatment. The aim of this study was to evaluate the RT-induced PD-L1 upregulation in the tumor and its microenvironment using immunoPET/CT imaging of two syngeneic murine tumor models (HPV+ head and neck squamous cell carcinoma (HNSCC) or B16F10 melanoma). Tumors were established in two locations per mouse (neck and flank), and fractionated RT (2 Gy × 4 or 2 Gy × 10) was delivered only to the neck tumor, alone or during anti-PD-1 mAb immunotherapy. PD-L1 expression was measured by PET/CT imaging using Zr-89 labeled anti-mouse PD-L1 mAb, and results were validated by flow cytometry. PET/CT imaging demonstrated significantly increased tracer uptake in irradiated neck tumors compared with non-irradiated flank tumors. Ex vivo analysis by biodistribution and flow cytometry validated PD-L1 upregulation specifically in irradiated tumors. In the HNSCC model, RT-induced PD-L1 upregulation was only observed after 2 Gy × 10 fractionated RT, while in the B16F10 model upregulation of PD-L1 occurred after 2 Gy × 4 fractionated RT. Fractionated RT, but not anti-PD-1 therapy, upregulated PD-L1 expression on tumor and infiltrating inflammatory cells in murine models, which could be non-invasively monitored by immunoPET/CT imaging using Zr-89 labeled anti-mouse PD-L1 mAb, and differentially identified anti-PD-1 responsive as well as selectively irradiated tumors in vivo.

Fluorescence Imaging Topography Scanning System for intraoperative multimodal imaging.

Fluorescence imaging is a powerful technique with diverse applications in intraoperative settings. Visualization of three dimensional (3D) structures and depth assessment of lesions, however, are oftentimes limited in planar fluorescence imaging systems. In this study, a novel Fluorescence Imaging Topography Scanning (FITS) system has been developed, which offers color reflectance imaging, fluorescence imaging and surface topography scanning capabilities. The system is compact and portable, and thus suitable for deployment in the operating room without disturbing the surgical flow. For system performance, parameters including near infrared fluorescence detection limit, contrast transfer functions and topography depth resolution were characterized. The developed system was tested in chicken tissues ex vivo with simulated tumors for intraoperative imaging. We subsequently conducted in vivo multimodal imaging of sentinel lymph nodes in mice using FITS and PET/CT. The PET/CT/optical multimodal images were co-registered and conveniently presented to users to guide surgeries. Our results show that the developed system can facilitate multimodal intraoperative imaging.

Tumor mitochondria-targeted photodynamic therapy with a translocator protein (TSPO)-specific photosensitizer.

Photodynamic therapy (PDT) has been proven to be a minimally invasive and effective therapeutic strategy for cancer treatment. It can be used alone or as a complement to conventional cancer treatments, such as surgical debulking and chemotherapy. The mitochondrion is an attractive target for developing novel PDT agents, as it produces energy for cells and regulates apoptosis. Current strategy of mitochondria targeting is mainly focused on utilizing cationic photosensitizers that bind to the negatively charged mitochondria membrane. However, such an approach is lack of selectivity of tumor cells. To minimize the damage on healthy tissues and improve therapeutic efficacy, an alternative targeting strategy with high tumor specificity is in critical need. Herein, we report a tumor mitochondria-specific PDT agent, IR700DX-6T, which targets the 18kDa mitochondrial translocator protein (TSPO). IR700DX-6T induced apoptotic cell death in TSPO-positive breast cancer cells (MDA-MB-231) but not TSPO-negative breast cancer cells (MCF-7). In vivo PDT study suggested that IR700DX-6T-mediated PDT significantly inhibited the growth of MDA-MB-231 tumors in a target-specific manner. These combined data suggest that this new TSPO-targeted photosensitizer has great potential in cancer treatment. STATEMENT OF SIGNIFICANCE: Photodynamic therapy (PDT) is an effective and minimally invasive therapeutic technique for treating cancers. Mitochondrion is an attractive target for developing novel PDT agents, as it produces energy to cells and regulates apoptosis. Current mitochondria targeted photosensitizers (PSs) are based on cationic molecules, which interact with the negatively charged mitochondria membrane. However, such PSs are not specific for cancerous cells, which may result in unwanted side effects. In this study, we developed a tumor mitochondria-targeted PS, IR700DX-6T, which binds to translocator protein (TSPO). This agent effectively induced apoptosis in TSPO-positive cancer cells and significantly inhibited tumor growth in TSPO-positive tumor-bearing mice. These combined data suggest that IR700DX-6T could become a powerful tool in the treatment of multiple cancers that upregulate TSPO.

Selection and characterization of high affinity VEGFR1 antibodies from a novel human binary code scFv phage library.

VEGFR1 is a receptor tyrosine kinase that has been implicated in cancer pathogenesis. It is upregulated in angiogenic endothelial cells and expressed on human tumor cells as well. VEGFR1 positive hematopoietic progenitor cells home to sites of distant metastases prior to the arrival of the tumor cells thus establishing a pre-metastatic niche. To discover high affinity human antibodies selective for VEGFR1 molecular imaging or for molecularly targeted therapy, a novel phage display scFv library was assembled and characterized. The library was constructed from the humanized 4D5 framework that was mostly comprised tyrosine and serine residues in four complimentary determining regions (CDRs). The library produced diverse and functional antibodies against a panel of proteins, some of which are of biomedical interest including, CD44, VEGFA, and VEGFR1. After panning, these antibodies had affinity strong enough for molecular imaging or targeted drug delivery without the need for affinity maturation. One of the anti-VEGFR1 scFvs recognized its cognate receptor and was selective for the VEGFR1.

Utilizing the Multiradionuclide Resolving Power of SPECT and Dual Radiolabeled Single Molecules to Assess Treatment Response of Tumors.

PURPOSE: Single photon emission computed tomography (SPECT) radionuclide pairs having distinct decay rates and different energy maxima enable simultaneous detection of dual gamma signals and real-time assessment of dynamic functional and molecular processes in vivo. Here, we report image acquisition and quantification protocols for a single molecule labeled with two different radionuclides for functional SPECT imaging. PROCEDURES: LS370 and LS734 were prepared using modular solid phase peptide synthesis. Each agent has a caspase-3 cleavable reporting motif, flanked by a tyrosine residue and a chelator at the opposite end of molecule. Cell uptake and efflux were assessed in human MDA-MB-231 breast cancer cells. Biodistribution studies were conducted in tumor naive and orthotopic 4T1 metastatic breast cancer tumor mice. NanoSPECT dual-imaging validation and attenuation correction parameters were developed using phantom vials containing varying radionuclide concentrations. Proof-of-principle SPECT imaging was performed in MMTV-PyMT transgenic mice. RESULTS: LS370 and LS734 were singly or dually radiolabeled with (125)I and (111)In or (99m)Tc. Cell assays demonstrated 11-fold higher percent uptake (P < 0.001) of [(125)I]LS734 (3.6 ± 0.5) compared to [(125)I]LS370 (0.3 ± 0.3) at 2 h. Following chemotherapy, cellular retention of [(125)I]LS734 was 3-fold higher (P < 0.05) than untreated cells. Pharmacokinetics at 1 h postinjection demonstrated longer blood retention (%ID/g) for [(125)I]LS734 (3.2 ± 0.9) compared to [(125)I]LS370 (1.6 ± 0.1). In mice bearing bilateral orthotopic 4T1 tumors, the uptake (%ID/g) was 2.4 ± 0.3 for [(125)I]LS734 and 1.2 ± 0.03 for [(125)I]LS370. The iodinated tyrosine peptide residue label was stable under in vitro conditions for up to 24 h; rapid systemic deiodination (high thyroid uptake) was observed in vivo. Phantom studies using standards demonstrated deconvolution of radionuclide signals based on different gamma ray energies. In MMTV-PyMT mice imaged with dual-labeled [(111)In]-[(125)I]LS734, the gamma signals were separable and quantifiable. CONCLUSIONS: Image processing protocols were developed for quantitative signal separation resulting from a caspase-3 responsive dual-radiolabeled SPECT probe. Crosstalk unmixing was obtained for multiradionuclide NanoSPECT imaging. In vitro and in vivo data demonstrated structure-activity relationships for developing functional agents for ratiometric SPECT imaging.

Folate receptor-targeted multimodality imaging of ovarian cancer in a novel syngeneic mouse model.

A new transplantable ovarian tumor model is presented using a novel folate receptor (FR) positive, murine ovarian cancer cell line that emulates the human disease and induces widespread intraperitoneal (i.p.) tumors in immunocompetent mice within 4-8 weeks of implantation. Tumor development was monitored using a new positron emission tomography (PET) FR-targeting reporter with PET/computerized tomography (PET/CT) and fluorescence molecular tomography (FMT) using a commercial FR-targeting reporter. Conventional structural magnetic resonance imaging (MRI) was also performed. Adult female C57BL/6 mice were injected i.p. with 6 × 10(6) MKP-L FR+ cells. Imaging was performed weekly beginning 2 weeks after tumor induction. The albumin-binding, FR-targeting ligand cm09 was radiolabeled with the positron emitter (68)Ga and used to image the tumors with a small animal PET/CT. The FR-reporter FolateRSense 680 (PerkinElmer) was used for FMT and flow cytometry. Preclinical MRI (7 T) without FR-targeting was compared with the PET and FMT molecular imaging. Tumors were visible by all three imaging modalities. PET/CT had the highest imaging sensitivity at 3-3.5 h postadministration (mean %IA/g mean > 6) and visualized tumors earlier than the other two modalities with lower kidney uptake (mean %IA/g mean < 17) than previously reported FR-targeting agents in late stage disease. FMT showed relatively low FR-targeted agent in the bladder and kidneys, but yielded the lowest anatomical image resolution. MRI produced the highest resolution images, but it was difficult to distinguish tumors from abdominal organs during early progression since a FR-targeting MRI reporter was not used. Nevertheless, there was good correlation of imaging biomarkers between the three modalities. Tumors in the mouse ovarian cancer model could be detected using FR-targeted imaging as early as 2 weeks post i.p. injection of tumor cells. An imaging protocol should combine one or more of the modalities, e.g., PET/CT or PET/MRI for optimal tumor detection and delineation from surrounding tissues.

Near-infrared triple-helical peptide with quenched fluorophores for optical imaging of MMP-2 and MMP-9 proteolytic activity in vivo.

The gelatinase members of the MMP family have consistently been associated with tumor invasiveness, which make them an attractive target for molecular imaging. We report new activatable proteolytic optical imaging agents that consist of triple-helical peptide (THP) conjugates, with high specificity to the gelatinases, bearing quenched cypate dyes. With quenching efficiencies up to 51%, the amplified fluorescence signal upon cypate3-THP hydrolysis by the gelatinases (kcat/KM values of 6.4×10(3) M(-1) s(-1) to 9.1×10(3) M(-1) s(-1) for MMP-2 and MMP-9, respectively) in mice bearing human fibrosarcoma xenografted tumors was monitored with fluorescence molecular tomography. There was significant fluorescence enhancement within the tumor and this enhancement was reduced by treatment with pan-MMP inhibitor, Ilomastat. These data, combined with the gelatinase substrate specificity observed in vitro, indicated the observed fluorescence at the site of the tumor was due to gelatinase mediated hydrolysis of cypate3-THP.

Discovery of hapten-specific scFv from a phage display library and applications for HER2-positive tumor imaging.

In this study, an anti-hapten antibody (single chain Fv, scFv) against a hapten probe was developed as a unique reporter system for molecular imaging or therapy. The hapten peptide (histamine-succinyl-GSYK, Him) was synthesized for phage displayed scFv affinity selection and for conjugation with cypate (Cy-Him) for in vivo near-infrared (NIR) optical imaging. Hapten-specific scFvs were affinity selected from the human single fold phage display scFv libraries (Tomlinson I + J) with high specificity and affinity. Utilizing HER2 targeting as a model system, the highest affinity scFv (clone J42) was recombinantly fused to an anti-HER2 affibody (scFv-L-Aff) with no loss of affinity of either protein. The functionality of the hapten-scFv reporter system was tested in vitro with a HER2-positive human breast cancer cell line, SK-BR3, and in vivo with SK-BR3 xenografts. ScFv-L-Aff mediated the binding of the hapten to HER2 on SK-BR3 cells and from tissue from the SK-BR3 xenograft; however, scFv-L-Aff did not mediate uptake of the hapten in the SK-BR3 xenografted tumors, presumably due to rapid internalization of the HER2/scFv-L-Aff complex. Our results suggest that this hapten-peptide and anti-hapten scFv can be a universal reporter system in a wide range of imaging and therapeutic applications.

Evaluation of phage display discovered peptides as ligands for prostate-specific membrane antigen (PSMA).

The aim of this study was to identify potential ligands of PSMA suitable for further development as novel PSMA-targeted peptides using phage display technology. The human PSMA protein was immobilized as a target followed by incubation with a 15-mer phage display random peptide library. After one round of prescreening and two rounds of screening, high-stringency screening at the third round of panning was performed to identify the highest affinity binders. Phages which had a specific binding activity to PSMA in human prostate cancer cells were isolated and the DNA corresponding to the 15-mers were sequenced to provide three consensus sequences: GDHSPFT, SHFSVGS and EVPRLSLLAVFL as well as other sequences that did not display consensus. Two of the peptide sequences deduced from DNA sequencing of binding phages, SHSFSVGSGDHSPFT and GRFLTGGTGRLLRIS were labeled with 5-carboxyfluorescein and shown to bind and co-internalize with PSMA on human prostate cancer cells by fluorescence microscopy. The high stringency requirements yielded peptides with affinities KD~1 µM or greater which are suitable starting points for affinity maturation. While these values were less than anticipated, the high stringency did yield peptide sequences that apparently bound to different surfaces on PSMA. These peptide sequences could be the basis for further development of peptides for prostate cancer tumor imaging and therapy.

Multimodal fluorescence-mediated tomography and SPECT/CT for small-animal imaging.

UNLABELLED: Spatial and temporal coregistration of nuclear and optical images can enable the fusion of the information from these complementary molecular imaging modalities. A critical challenge is in integrating the optical and nuclear imaging hardware. Flexible fiber-based fluorescence-mediated tomography (FMT) systems provide a viable solution. The various bore sizes of small-animal nuclear imaging systems can potentially accommodate the FMT fiber imaging arrays. In addition, FMT imaging facilitates coregistration of the nuclear and optical contrasts in time. Herein, we combine a fiber-based FMT system with a preclinical SPECT/CT platform. Feasibility of in vivo imaging is demonstrated by tracking a monomolecular multimodal imaging agent (MOMIA) during transport from the forepaw to the axillary lymph node region of a rat. METHODS: The fiber-based, video-rate FMT imaging system is composed of 12 sources (785- and 830-nm laser diodes) and 13 detectors. To maintain high temporal sampling, the system simultaneously acquires ratio-metric data at each detector. A 3-dimensional finite element model derived from CT projections provides anatomically based light propagation modeling. Injection of a MOMIA intradermally into the forepaw of rats provided spatially and temporally coregistered nuclear and optical contrasts. FMT data were acquired concurrently with SPECT and CT data. The incorporation of SPECT data as a priori information in the reconstruction of FMT data integrated both optical and nuclear contrasts. RESULTS: Accurate depth localization of phantoms with different thicknesses was accomplished with an average center-of-mass error of 4.1 ± 2.1 mm between FMT and SPECT measurements. During in vivo tests, fluorescence and radioactivity from the MOMIA were colocalized in spatially coincident regions with an average center-of-mass error of 2.68 ± 1.0 mm between FMT and SPECT for axillary lymph node localization. Intravital imaging with surgical exposure of the lymph node validated the localization of the optical contrast. CONCLUSION: The feasibility of integrating a fiber-based, video-rate FMT system with a commercial preclinical SPECT/CT platform was established. These coregistered FMT and SPECT/CT results with MOMIAs may facilitate the development of the next generation of preclinical and clinical multimodal optical-nuclear platforms for a broad array of imaging applications and help elucidate the underlying biologic processes relevant to cancer diagnosis and therapy monitoring.

Multimodal sentinel lymph node mapping with single-photon emission computed tomography (SPECT)/computed tomography (CT) and photoacoustic tomography.

The identification of cancer cells in the lymph nodes surrounding a tumor is important in establishing a prognosis. Optical detection techniques such as fluorescence and photoacoustic tomography (PAT) have been reported in preclinical studies for noninvasive sentinel lymph node (SLN) mapping. A method for validation of these techniques is needed for clinical trials. We report the use of a multimodal optical-radionuclear contrast agent as a validation tool for PAT in a preclinical model. Methylene blue (MB) was radiolabeled with (125)I for multimodal SLN mapping and used in conjunction with MB to assess the feasibility of multimodal SLN mapping in a rat model by PAT and single-photon emission computed tomography (SPECT). MB provided sufficient contrast for identifying SLNs noninvasively with a PAT system adapted from a clinical ultrasound imaging system. The signal location was corroborated by SPECT using (125)I labeled MB. The translation of PAT into the clinic can be facilitated by a direct comparison with established imaging methods using a clinically relevant dual SPECT and photoacoustic imaging agent. The new high-resolution PAT is a promising technology for the sensitive and accurate SLN detection in cancer patients.

Detection of MMP-2 and MMP-9 activity in vivo with a triple-helical peptide optical probe.

We report a novel activatable NIR fluorescent probe for in vivo detection of cancer-related matrix metalloproteinase (MMP) activity. The probe is based on a triple-helical peptide substrate (THP) with high specificity for MMP-2 and MMP-9 relative to other members of the MMP family. MMP-2 and MMP-9 (also known as gelatinases) are specifically associated with cancer cell invasion and cancer-related angiogenesis. At the center of each 5 kDa peptide strand is a gelatinase sensitive sequence flanked by 2 Lys residues conjugated with NIR fluorescent dyes. Upon self-assembly of the triple-helical structure, the 3 peptide chains intertwine, bringing the fluorophores into close proximity and reducing fluorescence via quenching. Upon enzymatic cleavage of the triple-helical peptide, 6 labeled peptide chains are released, resulting in an amplified fluorescent signal. The fluorescence yield of the probe increases 3.8-fold upon activation. Kinetic analysis showed a rate of LS276-THP hydrolysis by MMP-2 (k(cat)/K(M) = 30,000 s(-1) M(-1)) similar to that of MMP-2 catalysis of an analogous fluorogenic THP. Administration of LS276-THP to mice bearing a human fibrosarcoma xenografted tumor resulted in a tumor fluorescence signal more than 5-fold greater than that of muscle. This signal enhancement was reduced by treatment with the MMP inhibitor Ilomostat, indicating that the observed tumor fluorescence was indeed enzyme mediated. These results are the first to demonstrate that triple-helical peptides are suitable for highly specific in vivo detection of tumor-related MMP-2 and MMP-9 activity.

Detection of enzyme activity in orthotopic murine breast cancer by fluorescence lifetime imaging using a fluorescence resonance energy transfer-based molecular probe.

Cancer-related enzyme activity can be detected noninvasively using activatable fluorescent molecular probes. In contrast to "always-on" fluorescent molecular probes, activatable probes are relatively nonfluorescent at the time of administration due to intramolecular fluorescence resonance energy transfer (FRET). Enzyme-mediated hydrolysis of peptide linkers results in reduced FRET and increase of fluorescence yield. Separation of signal from active and inactive probe can be difficult with conventional intensity-based fluorescence imaging. Fluorescence lifetime (FLT) measurement is an alternative method to detect changes in FRET. Thus, we investigate FLT imaging for in vivo detection of FRET-based molecular probe activation in an orthotopic breast cancer model. Indeed, the measured FLT of the enzyme-activatable molecular probe increases from 0.62 ns just after injection to 0.78 ns in tumor tissue after 4 h. A significant increase in FLT is not observed for an always-on targeted molecular probe with the same fluorescent reporter. These results show that FLT contrast is a powerful addition to preclinical imaging because it can report molecular activity in vivo due to changes in FRET. Fluorescence lifetime imaging exploits unique characteristics of fluorescent molecular probes that can be further translated into clinical applications, including noninvasive detection of cancer-related enzyme activity.