Imaging Early-Stage Metastases Using an 18F-Labeled VEGFR-1-Specific Single Chain VEGF Mutant.

PURPOSE: Metastatic breast cancer is the second leading cause of cancer-related death in women. The 5-year survival rate for metastatic breast cancer has remained near 26.9 % for over a decade. The recruitment of hematopoietic stem cells with high expression of the vascular endothelial growth factor receptor 1 (VEGFR-1) has been implicated in early stages of metastasis formation. We propose the use of an 18F-labeled single-chain version of VEGF121, re-engineered to be selective for VEGFR-1 (scVR1), as a positron emission tomography (PET) imaging agent to non-invasively image early-stage metastases. PROCEDURES: scVR1 was 18F-labeled via a biorthogonal click reaction between site-specifically trans-cyclooctene functionalized scVR1 and an Al18F labeled tetrazine-NODA (1,4,7-triazacyclononane-1,4-diiacetic acid). The [18F]AlF-NODA-scVR1 was purified using a PD10 column and subsequently analyzed on HPLC to determine radiochemical purity. Animal experiments were performed in 6-8-week-old female BALB/c mice bearing orthotopic primary 4T1 breast tumors or 4T1 metastatic lesions. The [18F]AlF-NODA-scVR1 tracer was administered via tail vein injection; PET imaging and ex vivo analysis was performed 2 h post-injection. RESULTS: The [18F]AlF-NODA-scVR1 was prepared with a 98.2 ± 1.5 % radiochemical purity and an apparent molar activity of 7.5 ± 1.2 GBq/µmol. The specific binding of scVR1 to VEGFR-1 was confirmed via bead-based assay. The ex vivo biodistribution showed tumor uptake of 3.5 ± 0.5 % ID/g and was readily observable in PET images. Metastasis formation was detected with [18F]AlF-NODA-scVR1 tracer showing colocalization with bioluminescent imaging as well as ex vivo autoradiography and immunofluorescent staining of VEGFR-1. CONCLUSIONS: The diagnostic capabilities of the [18F]AlF-NODA-scVR1 PET tracer was confirmed in both orthotopic and metastatic murine cancer models. These results support the potential use of [18F]AlF-NODA-scVR1 as a PET tracer that could image metastases, providing clinicians with an additional tool to assess a patient's need for adjuvant therapies.

Targeting Fluorescent Nanodiamonds to Vascular Endothelial Growth Factor Receptors in Tumor.

The increased expression of vascular endothelial growth factor (VEGF) and its receptors is associated with angiogenesis in a growing tumor, presenting potential targets for tumor-selective imaging by way of targeted tracers. Though fluorescent tracers are used for targeted in vivo imaging, the lack of photostability and biocompatibility of many current fluorophores hinder their use in several applications involving long-term, continuous imaging. To address these problems, fluorescent nanodiamonds (FNDs), which exhibit infinite photostability and excellent biocompatibility, were explored as fluorophores in tracers for targeting VEGF receptors in growing tumors. To explore FND utility for imaging tumor VEGF receptors, we used click-chemistry to conjugate multiple copies of an engineered single-chain version of VEGF site-specifically derivatized with trans-cyclooctene (scVEGF-TCO) to 140 nm FND. The resulting targeting conjugates, FND-scVEGF, were then tested for functional activity of the scVEGF moieties through biochemical and tissue culture experiments and for selective tumor uptake in Balb/c mice with induced 4T1 carcinoma. We found that FND-scVEGF conjugates retain high affinity to VEGF receptors in cell culture experiments and observed preferential accumulation of FND-scVEGF in tumors relative to untargeted FND. Microspectroscopy provided unambiguous determination of FND within tissue by way of the unique spectral shape of nitrogen-vacancy induced fluorescence. These results validate and invite the use of targeted FND for diagnostic imaging and encourage further optimization of FND for fluorescence brightness.

A "Dock and Lock" Approach to Preparation of Targeted Liposomes.

We developed a strategy for covalent coupling of targeting proteins to liposomes decorated with a standard adapter protein. This strategy is based on "dock and lock" interactions between two mutated fragments of human RNase I, a 1-15 aa fragment with the R4C amino acid substitution (Cys-tag), and a 21-127-aa fragment with the V118C substitution, (Ad-C). Upon binding to each other, Cys-tag and Ad-C spontaneously form a disulfide bond between the complementary 4C and 118C residues. Therefore, any targeting protein expressed with Cys-tag can be easily coupled to liposomes decorated with Ad-C. Here we describe the preparation of Ad-liposomes followed by coupling them to two Cys-tagged targeted proteins, human vascular endothelial growth factor expressed with N-terminal Cys-tag and a 254-aa long N-terminal fragment of anthrax lethal factor carrying C-terminal Cys-tag. Both proteins retain functional activity after coupling to Ad-C-decorated drug-loaded liposomes. We expect that our "dock and lock" strategy will open new opportunities for development of targeted therapeutic liposomes for research and clinical use.

Selective Imaging of VEGFR-1 and VEGFR-2 Using 89Zr-Labeled Single-Chain VEGF Mutants.

Vascular endothelial growth factor-A (VEGF-A) acts via 2 vascular endothelial growth factor receptors, VEGFR-1 and VEGFR-2, that play important and distinct roles in tumor biology. We reasoned that selective imaging of these receptors could provide unique information for diagnostics and for monitoring and optimizing responses to anticancer therapy, including antiangiogenic therapy. Herein, we report the development of 2 first-in-class 89Zr-labeled PET tracers that enable the selective imaging of VEGFR-1 and VEGFR-2. METHODS: Functionally active mutants of scVEGF (an engineered single-chain version of pan-receptor VEGF-A with an N-terminal cysteine-containing tag for site-specific conjugation), named scVR1 and scVR2 with enhanced affinity to, respectively, VEGFR-1 and VEGFR-2, were constructed. Parental scVEGF and its receptor-specific mutants were site-specifically derivatized with the 89Zr chelator desferroxamine B via a 3.4-kDa PEG linker. 89Zr labeling of the desferroxamine B conjugates furnished scV/Zr, scVR1/Zr, and scVR2/Zr tracers with high radiochemical yield (>87%), high specific activity (≥9.8 MBq/nmol), and purity (>99%). Tracers were tested in an orthotopic breast cancer model using 4T1luc-bearing syngeneic BALB/c mice. For testing tracer specificity, tracers were coinjected with an excess of cold proteins of the same or opposite receptor specificity or pan-receptor scVEGF. PET imaging, biodistribution, and dosimetry studies in mice, as well as immunohistochemical analysis of harvested tumors, were performed. RESULTS: All tracers rapidly accumulated in orthotopic 4T1luc tumors, allowing for the successful PET imaging of the tumors as early as 2 h after injection. Blocking experiments with an excess of pan-receptor or receptor-specific cold proteins indicated that more than 80% of tracer tumor uptake is VEGFR-mediated, whereas uptake in all major organs is not affected by blocking within the margin of error. Critically, blocking experiments indicated that VEGFR-mediated tumor uptake of scVR1/Zr and scVR2/Zr was mediated exclusively by the corresponding receptor, VEGFR-1 or VEGFR-2, respectively. In contrast, uptake of pan-receptor scV/Zr was mediated by both VEGFR-1 and VEGFR-2 at an approximately 2:1 ratio. CONCLUSION: First-in-class selective PET tracers for imaging VEGFR-1 and VEGFR-2 were constructed and successfully validated in an orthotopic murine tumor model.

Targeted scVEGF/(177)Lu radiopharmaceutical inhibits growth of metastases and can be effectively combined with chemotherapy.

BACKGROUND: scVEGF/(177)Lu is a novel radiopharmaceutical targeted by recombinant single-chain (sc) derivative of vascular endothelial growth factor (VEGF) that binds to and is internalized by vascular endothelial growth factor receptors (VEGFR). scVEGF/(177)Lu potential as adjuvant and neoadjuvant anti-angiogenic therapy was assessed in metastatic and orthotopic mouse models of triple-negative breast cancer. METHODS: Metastatic lesions in Balb/c mice were established by intracardiac injection of luciferase-expressing 4T1luc mouse breast carcinoma cells. Mice with metastatic lesions received single intravenous (i.v.) injection of well-tolerated dose of scVEGF/(177)Lu (7.4 MBq/mouse) at day 8 after 4T1luc cell injection. Primary orthotopic breast tumors in immunodeficient mice were established by injecting luciferase-expressing MDA231luc human breast carcinoma cells into mammary fat pad. Tumor-bearing mice were treated with single injections of scVEGF/(177)Lu (7.4 MBq/mouse, i.v), or liposomal doxorubicin (Doxil, 1 mg doxorubicin per kg, i.v.), or with a combination of Doxil and scVEGF/(177)Lu given at the same doses, but two hours apart. "Cold" scVEGF-targeting conjugate was included in controls and in Doxil alone group. The effects of treatments were defined by bioluminescent imaging (BLI), computed tomography (CT), computed microtomography (microCT), measurements of primary tumor growth, and immunohistochemical analysis. RESULTS: In metastatic model, adjuvant treatment with scVEGF/(177)Lu decreased overall metastatic burden and improved survival. In orthotopic primary tumor model, a combination of Doxil and scVEGF/(177)Lu was more efficient in tumor growth inhibition than each treatment alone. scVEGF/(177)Lu treatment decreased immunostaining for VEGFR-1, VEGFR-2, and pro-tumorigenic M2-type macrophage marker CD206. CONCLUSIONS: Selective targeting of VEGFR with well-tolerated doses of scVEGF/(177)Lu is effective in metastatic and primary breast cancer models and can be combined with chemotherapy. As high level of VEGFR expression is a common feature in a variety of cancers, targeted delivery of (177)Lu for specific receptor-mediated uptake warrants further exploration.

Noninvasive Imaging of Liposomal Delivery of Superparamagnetic Iron Oxide Nanoparticles to Orthotopic Human Breast Tumor in Mice.

PURPOSE: Magnetic resonance imaging (MRI) is widely used for diagnostic imaging in preclinical studies and in clinical settings. Considering the intrinsic low sensitivity and poor specificity of standard MRI contrast agents, the enhanced delivery of MRI tracers into tumors is an important challenge to be addressed. This study was intended to investigate whether delivery of superparamagnetic iron oxide nanoparticles (SPIONs) can be enhanced by liposomal SPION formulations for either "passive" delivery into tumor via the enhanced permeability and retention (EPR) effect or "active" targeted delivery to tumor endothelium via the receptors for vascular endothelial growth factor (VEGFRs). METHODS: In vivo MRI of orthotopic MDA-MB-231 tumors was performed on a preclinical 9.4 T MRI scanner following intravenous administration of either free/non-targeted or targeted liposomal SPIONs. RESULTS: In vivo MRI study revealed that only the non-targeted liposomal formulation provided a statistically significant accumulation of SPIONs in the tumor at four hours post-injection. The EPR effect contributes to improved accumulation of liposomal SPIONs in tumors compared to the presumably more transient retention during the targeting of the tumor vasculature via VEGFRs. CONCLUSIONS: A non-targeted liposomal formulation of SPIONs could be the optimal option for MRI detection of breast tumors and for the development of therapeutic liposomes for MRI-guided therapy.

Imaging VEGF receptor expression to identify accelerated atherosclerosis.

BACKGROUND: The biology of the vulnerable plaque includes increased inflammation and rapid growth of vasa vasorum, processes that are associated with enhanced vascular endothelial growth factor (VEGF)/ imaging receptors for VEGF (VEGFR) signaling and are accelerated in diabetes. This study was designed to test the hypothesis that VEGFRs in atherosclerotic plaques with a SPECT tracer scVEGF-PEG-DOTA/(99m)Tc (scV/Tc) can identify accelerated atherosclerosis in diabetes. METHODS: Male apolipoprotein E null (ApoE(-/-)) mice (6 weeks of age) were made diabetic (n = 10) or left as non-diabetic (n = 13). At 26 to 28 weeks of age, 5 non-diabetic mice were injected with functionally inactivated scV/Tc (in-scV/Tc) that does not bind to VEGF receptors, while 8 non-diabetic and 10 diabetic mice were injected with scV/Tc. After blood pool clearance, at 3 to 4 h post-injection, mice were injected with CT contrast agent and underwent SPECT/CT imaging. From the scans, regions of interest (ROI) were drawn on serial transverse sections comprising the proximal aorta and the percentage of injected dose (%ID) in ROIs was calculated. At the completion of imaging, mice were euthanized, proximal aorta explanted for gamma well counting to determine the percentage of injected dose per gram (%ID/g) uptake and immunohistochemical characterization. RESULTS: The uptake of scV/Tc in the proximal aorta, calculated from SPECT/CT co-registered scans as %ID, was significantly higher in the diabetic mice (0.036 ± 0.017%ID) compared to non-diabetic mice (0.017 ± 0.005%ID; P < 0.01), as was uptake measured as %ID/g in harvested aorta, 1.81 ± 0.50%ID/g in the diabetic group vs. 0.98 ± 0.25%ID/g in the non-diabetic group (P < 0.01). The nonspecific uptake of in-scV/Tc in proximal aorta was significantly lower than the uptake of functionally active scV/Tc. Immunostaining of the atherosclerotic lesions showed higher expression of VEGFR-1 and VEGFR-2 in the diabetic mice. CONCLUSION: These initial results suggest that imaging VEGFR with scV/Tc shows promise as a non-invasive approach to identify accelerated atherosclerosis.

Enhanced fluorescence diffuse optical tomography with indocyanine green-encapsulating liposomes targeted to receptors for vascular endothelial growth factor in tumor vasculature.

To develop an indocyanine green (ICG) tracer with slower clearance kinetics, we explored ICG-encapsulating liposomes (Lip) in three different formulations: untargeted (Lip/ICG), targeted to vascular endothelial growth factor (VEGF) receptors (scVEGF-Lip/ICG) by the receptor-binding moiety single-chain VEGF (scVEGF), or decorated with inactivated scVEGF (inactive-Lip/ICG) that does not bind to VEGF receptors. Experiments were conducted with tumor-bearing mice that were placed in a scattering medium with tumors located at imaging depths of either 1.5 or 2.0 cm. Near-infrared fluorescence diffuse optical tomography that provides depth-resolved spatial distributions of fluorescence in tumor was used for the detection of postinjection fluorescent signals. All liposome-based tracers, as well as free ICG, were injected intravenously into mice in the amounts corresponding to 5 nmol of ICG/mouse, and the kinetics of increase and decrease of fluorescent signals in tumors were monitored. A signal from free ICG reached maximum at 15-min postinjection and then rapidly declined with t1/2 of ~20 min. The signals from untargeted Lip/ICG and inactive-Lip/ICG also reached maximum at 15-min postinjection, however, declined somewhat slower than free ICG with t1/2 of ~30 min. By contrast, a signal from targeted scVEGF-Lip/ICG grew slower than that of all other tracers, reaching maximum at 30-min postinjection and declined much slower than that of other tracers with t1/2 of ~90 min, providing a more extended observation window. Higher scVEGF-Lip/ICG tumor accumulation was further confirmed by the analysis of fluorescence on cryosections of tumors that were harvested from animals at 400 min after injection with different tracers.

Imaging key biomarkers of tumor angiogenesis.

Angiogenesis is a fundamental requirement for tumor growth and therefore it is a primary target for anti-cancer therapy. Molecular imaging of angiogenesis may provide novel opportunities for early diagnostic and for image-guided optimization and management of therapeutic regimens. Here we reviewed the advances in targeted imaging of key biomarkers of tumor angiogenesis, integrins and receptors for vascular endothelial growth factor (VEGF). Tracers for targeted imaging of these biomarkers in different imaging modalities are now reasonably well-developed and PET tracers for integrin imaging are currently in clinical trials. Molecular imaging of longitudinal responses to anti-angiogenic therapy in model tumor systems revealed a complex pattern of changes in targeted tracer accumulation in tumor, which reflects drug-induced tumor regression followed by vascular rebound. Further work will define the competitiveness of targeted imaging of key angiogenesis markers for early diagnostic and image-guided therapy.

Molecular imaging of vascular endothelial growth factor receptors in graft arteriosclerosis.

OBJECTIVE: Vascular endothelial growth factor (VEGF) signaling plays a key role in the pathogenesis of vascular remodeling, including graft arteriosclerosis. Graft arteriosclerosis is the major cause of late organ failure in cardiac transplantation. We used molecular near-infrared fluorescent imaging with an engineered Cy5.5-labeled single-chain VEGF tracer (scVEGF/Cy) to detect VEGF receptors and vascular remodeling in human coronary artery grafts by molecular imaging. METHODS AND RESULTS: VEGF receptor specificity of probe uptake was shown by flow cytometry in endothelial cells. In severe combined immunodeficiency mice, transplantation of human coronary artery segments into the aorta followed by adoptive transfer of allogeneic human peripheral blood mononuclear cells led to significant neointima formation in the grafts over a period of 4 weeks. Near-infrared fluorescent imaging of transplant recipients at 4 weeks demonstrated focal uptake of scVEGF/Cy in remodeling artery grafts. Uptake specificity was demonstrated using an inactive homolog of scVEGF/Cy. scVEGF/Cy uptake predominantly localized in the neointima of remodeling coronary arteries and correlated with VEGF receptor-1 but not VEGF receptor-2 expression. There was a significant correlation between scVEGF/Cy uptake and transplanted artery neointima area. CONCLUSIONS: Molecular imaging of VEGF receptors may provide a noninvasive tool for detection of graft arteriosclerosis in solid organ transplantation.

Targeted systemic radiotherapy with scVEGF/177Lu leads to sustained disruption of the tumor vasculature and intratumoral apoptosis.

UNLABELLED: Tumor vessels abundantly express receptors for vascular endothelial growth factor (VEGF), despite treatment with conventional or antiangiogenic drugs. We wished to determine whether the high levels of VEGF receptor (VEGFR) within the tumor vasculature could be leveraged for intracellular delivery of therapeutically significant doses of scVEGF/(177)Lu, a novel radiopharmaceutical based on a recombinant single-chain (sc) derivative of VEGF, in orthotopic breast cancer models. METHODS: scVEGF-PEG (polyethylene gycol)-DOTA conjugates containing 2.0-, 3.4-, or 5.0-kDa PEG linkers site-specifically conjugated to a cysteine-containing tag (Cys-tag) in scVEGF were radiolabeled with (177)Lu (scVEGF/(177)Lu) for in vivo studies. Human MDA231luc and mouse 4T1luc cell lines were injected orthotopically to establish breast carcinoma tumors in immunodeficient and immunocompetent hosts, respectively. The effects of scVEGF/(177)Lu were defined by analysis of changes in tumor growth and immunohistochemical staining for the endothelial markers CD31 and VEGFR-2 and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining for intratumoral apoptosis. RESULTS: Biodistribution assays and dosimetric calculations established that scVEGF/(177)Lu with a 3.4-kDa PEG linker delivered the highest dose of radiation to tumors (69.9 cGy/MBq/g of tissue) and the lowest dose to the kidneys (33.3 cGy/MBq/organ). Total doses below 40 MBq/mouse of scVEGF/(177)Lu did not affect renal function, and 3 divided doses of 6.3 MBq/mouse or a bolus dose of 18.9 MBq/mouse induced only transient lymphopenia and weight loss (<10% baseline weight). In mice with orthotopic mammary breast carcinoma, intravenous injections of well-tolerated bolus and fractionated doses of scVEGF/(177)Lu in the range from 6.3 to 18.9 MBq/mouse (25-76 MBq/m(2)) resulted in dose-dependent tumor growth inhibition. Immunohistochemical analysis of tumors at 4-5 wk after single injections of scVEGF/(177)Lu indicated dose-dependent regression of tumor vasculature and widespread intratumoral apoptosis. A single dose of 7.4 MBq/mouse of scVEGF/(177)Lu given before a course of bevacizumab or sunitinib treatment enhanced the antiangiogenic effects of both drugs. CONCLUSION: Selective targeting of VEGFR in tumor vasculature with well-tolerated doses of scVEGF/(177)Lu is effective in orthotopic breast cancer models. As high levels of VEGFR expression in the tumor vasculature are a common feature in a variety of cancers, targeting tumor angiogenesis with scVEGF/(177)Lu warrants further exploration.

Targeting the unfolded protein response in cancer therapy.

Rapid growth of tumor cells coupled with inadequate vascularization leads to shortage of oxygen and nutrients. The unfolded protein response (UPR), a defense cellular mechanism activated during such stress conditions, is a complex process that includes upregulation of the endoplasmic reticulum chaperones, such as glucose-regulated protein 78 (GRP78). Due to its central role in UPR, GRP78 is overexpressed in many cancers; it is implicated in cancer cell survival through supporting of drug- and radioresistance as well as metastatic dissemination, and is generally associated with poor outcome. This is the reason why selective destruction of GRP78 could become a novel anticancer strategy. GRP78 is the only known substrate of the proteolytic A subunit (SubA) of a bacterial AB(5) toxin, and the selective SubA-induced cleavage of GRP78 leads to massive cell death. Targeted delivery of SubA into cancer cells via specific receptor-mediated endocytosis could be a suitable strategy for assaulting tumor cells. We fused SubA to epidermal growth factor (EGF), whose receptor (EGFR) is frequently overexpressed in tumor cells, and demonstrated that the resulting EGF-SubA immunotoxin is an effective killer of EGFR-positive tumor cells. Furthermore, because of its unique mechanism of action, EGF-SubA synergizes with UPR-inducing drugs, which opens a possibility for the development of mechanism-based combination regimens for effective anticancer therapy. In this chapter, we provide experimental protocols for the assessment of the effects of EGF-SubA on EGFR-positive cancer cells, either alone or in combination with UPR-inducing drugs.

In vivo, dual-modality OCT/LIF imaging using a novel VEGF receptor-targeted NIR fluorescent probe in the AOM-treated mouse model.

PURPOSE: Increased vascular endothelial growth factor (VEGF) receptor expression has been found at the sites of angiogenesis, particularly in tumor growth areas, as compared with quiescent vasculature. An increase in VEGF receptor-2 is associated with colon cancer progression. The in vivo detection of VEGF receptor is of interest for the purposes of studying basic mechanisms of carcinogenesis, making clinical diagnoses, and monitoring the efficacy of chemopreventive and therapeutic agents. In this study, a novel single chain (sc)VEGF-based molecular probe is utilized in the azoxymethane (AOM)-treated mouse model of colorectal cancer to study delivery route and specificity for disease. PROCEDURES: The probe was constructed by site-specific conjugation of a near-infrared fluorescent dye, Cy5.5, to scVEGF and detected in vivo with a dual-modality optical coherence tomography/laser-induced fluorescence (OCT/LIF) endoscopic system. A probe inactivated via excessive biotinylation was utilized as a control for nonreceptor-mediated binding. The LIF excitation source was a 633-nm He:Ne laser, and red/near-infrared fluorescence was detected with a spectrometer. OCT was used to obtain two-dimensional longitudinal tomograms at eight rotations in the distal colon. Fluorescence emission levels were correlated with OCT-detected disease in vivo. OCT-detected disease was verified with hematoxylin and eosin stained histology slides ex vivo. RESULTS: High fluorescence emission intensity from the targeted probe was correlated with tumor presence as detected using OCT in vivo and VEGFR-2 immunostaining on histological sections ex vivo. The inactivated probe accumulated preferentially on the surface of tumor lesions and in lymphoid aggregate tissue and was less selective for VEGFR-2. CONCLUSION: The scVEGF/Cy probe delivered via colonic lavage reaches tumor vasculature and selectively accumulates in VEGFR-2-positive areas, resulting in high sensitivity and specificity for tumor detection. The combination of OCT and LIF imaging modalities may allow the simultaneous study of tumor morphology and protein expression for the development of diagnostic and therapeutic methods for colorectal cancer.

Specific targeting of tumor endothelial cells by a shiga-like toxin-vascular endothelial growth factor fusion protein as a novel treatment strategy for pancreatic cancer.

PURPOSE: Tumor endothelial cells express vascular endothelial growth factor receptor 2 (VEGFR-2). VEGF can direct toxins to tumor vessels through VEGFR-2 for antiangiogenic therapy. This study aimed to selectively damage the VEGFR-2-overexpressing vasculature of pancreatic cancer by SLT-VEGF fusion protein comprising VEGF and the A subunit of Shiga-like toxin which inhibits protein synthesis of cells with high VEGFR-2 expression. EXPERIMENTAL DESIGN: Expression of VEGF and VEGF receptors was evaluated in human pancreatic cancer cells (AsPC-1, HPAF-2) and in normal human endothelial cells (HUVEC) by reverse transcription-polymerase chain reaction. Cells were treated with SLT-VEGF (0.1-10 nM), and cell viability, proliferation, and endothelial tube formation were assessed. Orthotopic pancreatic cancer (AsPC-1, HPAF-2) was induced in nude mice. Animals were treated with SLT-VEGF fusion protein alone or in combination with gemcitabine. Treatment began 3 days or 6 weeks after tumor induction. Primary tumor volume and dissemination were determined after 14 weeks. Microvessel density and expression of VEGF and VEGF receptors were analyzed by immunohistochemistry. RESULTS: SLT-VEGF did not influence proliferation of pancreatic cancer cells; HUVECs (low-level VEGFR-2) reduced their proliferation rate and tube formation but not their viability. SLT-VEGF fusion protein reduced tumor growth and dissemination, increasing 14-week survival (AsPC-1, up to 75%; HPAF-2, up to 83%). Results of gemcitabine were comparable with SLT-VEGF monotherapy. Combination partly increased the therapeutic effects in comparison to the respective monotherapies. Microvessel density was reduced in all groups. Intratumoral VEGFR-2 expression was found in endothelial but not in tumor cells. CONCLUSIONS: SLT-VEGF is toxic for tumor vasculature rather than for normal endothelial or pancreatic cancer cells. SLT-VEGF treatment in combination with gemcitabine may provide a novel approach for pancreatic cancer.

Fluorescence imaging of vascular endothelial growth factor in tumors for mice embedded in a turbid medium.

We demonstrate the feasibility of fluorescence imaging of deeply seated tumors using mice injected with an angiogenesis tracer, a vascular endothelial growth factor conjugated with the infrared dye cyanine 7 (VEGF/Cy7). Our optical-only imaging reconstruction method separately estimates the target depth, and then applies this information to reconstruct functional information such as fluorophore concentration. Fluorescence targets with concentrations as low as sub-25 nM are well reconstructed at depths up to 2 cm in both homogeneous and heterogeneous media with this technique.

Noninvasive assessment of tumor VEGF receptors in response to treatment with pazopanib: a molecular imaging study.

Vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) drive angiogenesis, and several VEGFR inhibitors are already approved for use as single agents or in combination with chemotherapy. Although there is a clear benefit with these drugs in a variety of tumors, the clinical response varies markedly among individuals. Therefore, there is a need for an efficient method to identify patients who are likely to respond to antiangiogenic therapy and to monitor its effects over time. We have recently developed a molecular imaging tracer for imaging VEGFRs known as scVEGF/(99m)Tc; an engineered single-chain (sc) form of VEGF radiolabeled with technetium Tc 99m ((99m)Tc). After intravenous injection, scVEGF/(99m)Tc preferentially binds to and is internalized by VEGFRs expressed within tumor vasculature, providing information on prevalence of functionally active receptors. We now report that VEGFR imaging readily detects the effects of pazopanib, a small-molecule tyrosine kinase inhibitor under clinical development, which selectively targets VEGFR, PDGFR, and c-Kit in mice with HT29 tumor xenografts. Immunohistochemical analysis confirmed that the changes in VEGFR imaging reflect a dramatic pazopanib-induced decrease in the number of VEGFR-2(+)/CD31(+) endothelial cells (ECs) within the tumor vasculature followed by a relative increase in the number of ECs at the tumor edges. We suggest that VEGFR imaging can be used for the identification of patients that are responding to VEGFR-targeted therapies and for guidance in rational design, dosing, and schedules for combination regimens of antiangiogenic treatment.

A "dock and lock" approach to preparation of targeted liposomes.

We developed a strategy for covalent coupling of targeting proteins to liposomes decorated with a standard adapter protein. This strategy is based on "dock and lock" the interactions between two mutated fragments of human RNase I, a 1-15-aa fragment with the R4C amino acid substitution, (Cys-tag), and a 21-127-aa fragment with the V118C substitution, (Ad-C). Upon binding to each other, Cys-tag and Ad-C spontaneously form a disulfide bond between the complimentary 4C and 118C residues. Therefore, any targeting protein expressed with Cys-tag can be easily coupled to liposomes decorated with Ad-C. Here, we describe the preparation of Ad-liposomes followed by coupling them to two Cys-tagged targeted proteins, human vascular endothelial growth factor expressed with N-terminal Cys-tag, and a 254-aa long N-terminal fragment of anthrax lethal factor carrying C-terminal Cys-tag. Both proteins retain functional activity after coupling to Ad-C-decorated drug-loaded liposomes. We expect that our "dock and lock" strategy will open new opportunities for development of targeted therapeutic liposomes for research and clinical use.

Chaperone-targeting cytotoxin and endoplasmic reticulum stress-inducing drug synergize to kill cancer cells.

Diverse physiological and therapeutic insults that increase the amount of unfolded or misfolded proteins in the endoplasmic reticulum (ER) induce the unfolded protein response, an evolutionarily conserved protective mechanism that manages ER stress. Glucose-regulated protein 78/immunoglobulin heavy-chain binding protein (GRP78/BiP) is an ER-resident protein that plays a central role in the ER stress response and is the only known substrate of the proteolytic A subunit (SubA) of a novel bacterial AB(5) toxin. Here, we report that an engineered fusion protein, epidermal growth factor (EGF)-SubA, combining EGF and SubA, is highly toxic to growing and confluent epidermal growth factor receptor-expressing cancer cells, and its cytotoxicity is mediated by a remarkably rapid cleavage of GRP78/BiP. Systemic delivery of EGF-SubA results in a significant inhibition of human breast and prostate tumor xenografts in mouse models. Furthermore, EGF-SubA dramatically increases the sensitivity of cancer cells to the ER stress-inducing drug thapsigargin, and vice versa, demonstrating the first example of mechanism-based synergism in the action of a cytotoxin and an ER-targeting drug.

Inhibition of vascular endothelial growth factor receptor signaling in angiogenic tumor vasculature.

Neovascularization takes place in a large number of pathologies, including cancer. Significant effort has been invested in the development of agents that can inhibit this process, and an increasing number of such agents, known as antiangiogenic drugs, are entering clinical trials or being approved for clinical use. The key players involved in the development and maintenance of tumor neovasculature are vascular endothelial growth factor (VEGF) and its receptors (VEGFRs), and therefore VEGF/VEGFR signaling pathways have been a focus of anticancer therapies for several decades. This review focuses on two main approaches designed to selectively target VEGFRs, inhibiting VEGFR with small molecule inhibitors of receptor tyrosine kinase activity and inhibiting the binding of VEGF to VEGFRs with specific antibodies or soluble decoy VEGF receptors. The major problem with these strategies is that they appeared to be effective only in relatively small and unpredictable subsets of patients. An alternative approach would be to subvert VEGFR for intracellular delivery of cytotoxic molecules. We describe here one such molecule, SLT-VEGF, a fusion protein containing VEGF121 and the highly cytotoxic catalytic subunit of Shiga-like toxin.

Analysis of in situ and ex vivo vascular endothelial growth factor receptor expression during experimental aortic aneurysm progression.

OBJECTIVE: Mural inflammation and neovascularization are characteristic pathological features of abdominal aortic aneurysm (AAA) disease. Vascular endothelial growth factor receptor (VEGFR) expression may also mediate AAA growth and rupture. We examined VEGFR expression as a function of AAA disease progression in the Apolipoprotein E-deficient (Apo E(-/-)) murine AAA model. METHODS AND RESULTS: Apo E(-/-) mice maintained on a high-fat diet underwent continuous infusion with angiotensin II at 1000 ng/kg/min (Ang II) or vehicle (Control) via subcutaneous osmotic pump. Serial transabdominal ultrasound measurements of abdominal aortic diameter were recorded (n=16 mice, 3 to 4 time points per mouse) for up to 28 days. Near-infrared receptor fluorescent (NIRF) imaging was performed on Ang II mice (n=9) and Controls (n=5) with scVEGF/Cy, a single-chain VEGF homo-dimer labeled with Cy 5.5 fluorescent tracer (7 to 18 microg/mouse IV). NIRF with inactivated single chain VEGF/Cy tracer (scVEGF/In, 18 microg/mouse IV) was performed on 2 additional Ang II mice to control for nonreceptor-mediated tracer binding and uptake. After image acquisition and sacrifice, aortae were harvested for analysis. An additional AAA mouse cohort received either an oral angiogenesis inhibitor or suitable negative or positive controls to clarify the significance of angiogenesis in experimental aneurysm progression. Aneurysms developed in the suprarenal aortic segment of all Ang II mice. Significantly greater fluorescent signal was obtained from aneurysmal aorta as compared to remote, uninvolved aortic segments in Ang II scVEGF/Cy mice or AAA in scVEGF/In mice or suprarenal aortic segments in Control mice. Signal intensity increased in a diameter-dependent fashion in aneurysmal segments. Immunostaining confirmed mural VEGFR-2 expression in medial smooth muscle cells. Treatment with an angiogenesis inhibitor attenuated AAA formation while decreasing mural macrophage infiltration and CD-31(+) cell density. CONCLUSIONS: Mural VEGFR expression, as determined by scVEGF/Cy fluorescent imaging and VEGFR-2 immunostaining, increases in experimental AAAs in a diameter-dependent fashion. Angiogenesis inhibition limits AAA progression. Clinical VEGFR expression imaging strategies, if feasible, may improve real-time monitoring of AAA disease progression and response to suppressive strategies.