High-Contrast Detection of Somatostatin Receptor Subtype-2 for Fluorescence-Guided Surgery.

Dye design can influence the ability of fluorescently labeled imaging agents to generate tumor contrast and has become an area of significant interest in the field of fluorescence-guided surgery (FGS). Here, we show that the charge-balanced near-infrared fluorescent (NIRF) dye FNIR-Tag enhances the imaging properties of a fluorescently labeled somatostatin analogue. In vitro studies showed that the optimized fluorescent conjugate MMC(FNIR-Tag)-TOC bound primarily via somatostatin receptor subtype-2 (SSTR2), whereas its negatively charged counterpart with IRDye 800CW had higher off-target binding. NIRF imaging in cell line- and patient-derived xenograft models revealed markedly higher tumor contrast with MMC(FNIR-Tag)-TOC, which was attributed to increased tumor specificity. Ex vivo staining of surgical biospecimens from primary and metastatic tumors, as well as involved lymph nodes, demonstrated binding to human tumors. Finally, in an orthotopic tumor model, a simulated clinical workflow highlighted our unique ability to use standard preoperative nuclear imaging for selecting patients likely to benefit from SSTR2-targeted FGS. Our findings demonstrate the translational potential of MMC(FNIR-Tag)-TOC for intraoperative imaging and suggest broad utility for using FNIR-Tag in fluorescent probe development.

Comparison of HER2-Targeted Antibodies for Fluorescence-Guided Surgery in Breast Cancer.

Background: Although therapeutic advances have led to enhanced survival in patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer, detection of residual disease remains challenging. Here, we examine two approved anti-HER2 monoclonal antibodies (mAbs), trastuzumab and pertuzumab, as potential candidates for the development of immunoconjugates for fluorescence-guided surgery (FGS). Methods: mAbs were conjugated to the near-infrared fluorescent (NIRF) dye, IRDye800, and for quantitative in vitro assessment, to the radiometal chelator, desferrioxamine, to enable dual labeling with 89Zr. In vitro binding was evaluated in HER2-overexpressing (BT474, SKBR3) and HER2-negative (MCF7) cell lines. BT474 and MCF7 xenografts were used for in vivo and ex vivo fluorescence imaging. Results: In vitro findings demonstrated HER2-mediated binding for both fluorescent immunoconjugates and were in agreement with radioligand assays using dual-labeled immunoconjugates. In vivo and ex vivo studies showed preferential accumulation of the fluorescently-labeled mAbs in tumors and similar tumor-to-background ratios. In vivo HER2 specificity was confirmed by immunohistochemical staining of resected tumors and normal tissues. Conclusions: We showed for the first time that fluorescent trastuzumab and pertuzumab immunoconjugates have similar NIRF imaging performance and demonstrated the possibility of performing HER2-targeted FGS with agents that possess distinct epitope specificity.

Evaluation of Anti-LGR5 Antibodies by ImmunoPET for Imaging Colorectal Tumors and Development of Antibody-Drug Conjugates.

Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is highly expressed in colorectal tumors and marks colon cancer stem cells that drive tumor growth and metastasis. Recently, we showed that LGR5 is a promising target for antibody-drug conjugate (ADC) therapy. However, it is important to identify LGR5-positive tumors that would respond to ADC treatment. Prior to drug conjugation, we evaluated two different anti-LGR5 monoclonal antibodies (mAbs), 8F2 and 9G5, using 89Zr-immunoPET to select the optimal mAb for ADC development and tumor imaging. Binding, specificity, and internalization were compared, and mAbs were prescreened as ADC candidates against colon cancer cells using secondary ADCs. Both mAbs demonstrated strong, specific binding in 293T-LGR5 cells but not 293T-vector cells. In DLD-1 colorectal cancer cells, which express high levels of LGR5, the mAbs rapidly internalized into lysosomes and promoted ADC-induced cytotoxicity, with 8F2 exhibiting slightly higher potency. No binding was detected in DLD-1-shLGR5 (LGR5 knockdown) cells. 89Zr-DFO-LGR5 mAbs were generated and shown to retain high affinity and LGR5-dependent uptake in vitro. PET/CT imaging of DLD-1 tumors was performed 5 days postinjection of 89Zr-DFO-LGR5 mAbs, and findings were consistent with biodistribution data, which showed significantly higher tumor uptake (%ID/g) for 89Zr-DFO-8F2 (17.9 ± 2.2) compared to 89Zr-DFO-9G5 (5.5 ± 1.2) and 89Zr-DFO-IgG (3.8 ± 1.0). No significant uptake was observed in DLD-1-shLGR5 tumors. This study identifies 8F2 as the optimal candidate for ADC development and provides initial evidence that 89Zr-DFO-LGR5 mAbs may be utilized to stratify tumors which would respond best to LGR5-targeted ADC therapy.

Somatostatin Receptor Subtype-2 Targeting System for Specific Delivery of Temozolomide.

Temozolomide (TMZ) is a DNA alkylating agent that produces objective responses in patients with neuroendocrine tumors (NETs) when the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) is inactivated. At high doses, TMZ therapy exhausts MGMT activity but also produces dose-limiting toxicities. To reduce off-target effects, we converted the clinically approved radiotracer 68Ga-DOTA-TOC into a peptide-drug conjugate (PDC) for targeted delivery of TMZ to somatostatin receptor subtype-2 (SSTR2)-positive tumor cells. We used an integrated radiolabeling strategy for direct quantitative assessment of receptor binding, pharmacokinetics, and tissue biodistribution. In vitro studies revealed selective binding to SSTR2-positive cells with high affinity (5.98 ± 0.96 nmol/L), internalization, receptor-dependent DNA damage, cytotoxicity, and MGMT depletion. Imaging and biodistribution analysis showed preferential accumulation of the PDC in receptor-positive tumors and high renal clearance. This study identified a trackable SSTR2-targeting system for TMZ delivery and utilizes a modular design that could be broadly applied in PDC development.

A translational blueprint for developing intraoperative imaging agents via radiopharmaceutical-guided drug design.

Cancer imaging is a rapidly evolving field due to the discovery of novel molecular targets and the availability of corresponding techniques to detect them with high precision, accuracy, and sensitivity. Nuclear medicine is the most widely used molecular imaging modality and has a growing toolkit of clinically used radiopharmaceuticals that enable whole-body tumor visualization, staging, and treatment monitoring for a variety of tumors in a non-invasive manner. The need for similar imaging capabilities in the operating room has led to the emergence of fluorescence-guided surgery (FGS) as a powerful technique that gives surgeons unprecedented ability to distinguish tumors from healthy tissues. While a variety of strategies have been used to develop contrast agents for FGS, the use of radiopharmaceuticals as models brings exceptional translational potential and has increasingly been explored. Here, we review strategies used to convert clinically used radiopharmaceuticals into fluorescent and multimodal counterparts. Unique preclinical and clinical capabilities stemming from radiopharmaceutical-based agent design are also discussed to illustrate the advantages of this approach.

Combined multiphoton microscopy and somatostatin receptor type 2 imaging of pancreatic neuroendocrine tumors.

Pancreatic neuroendocrine tumors (PNETs) are a rare but increasingly more prevalent cancer with heterogeneous clinical and pathological presentation. Surgery is the preferred treatment for all hormone-expressing PNETs and any PNET greater than 2 cm, but difficulties arise when tumors are multifocal, metastatic, or small in size due to lack of effective surgical localization. Existing techniques such as intraoperative ultrasound provide poor contrast and resolution, resulting in low sensitivity for such tumors. Somatostatin receptor type 2 (SSTR2) is commonly overexpressed in PNETs and presents an avenue for targeted tumor localization. SSTR2 is often used for pre-operative imaging and therapeutic treatment, with recent studies demonstrating that somatostatin receptor imaging (SRI) can be applied in radioguided surgery to aid in removal of metastatic lymph nodes and achieving negative surgical margins. However not all PNETs express SSTR2, indicating labeled SRI could benefit from using a supplemental label-free technique such as multiphoton microscopy (MPM), which has proven useful in improving the accuracy of diagnosing more common exocrine pancreatic cancers. Our work tests the suitability of combined SRI and MPM for localizing PNETs by imaging and comparing samples of PNETs and normal pancreatic tissue. Specimens were labeled with a novel SSTR2-targeted contrast agent and imaged using fluorescence microscopy, and subsequently imaged using MPM to collect four autofluorescent channels and second harmonic generation. Our results show that a combination of both SRI and MPM provides enhanced contrast and sensitivity for localizing diseased tissue, suggesting that this approach could be a valuable clinical tool for surgical localization and treatment of PNETs.

Targeted Dual-Modal PET/SPECT-NIR Imaging: From Building Blocks and Construction Strategies to Applications.

Molecular imaging is an emerging non-invasive method to qualitatively and quantitively visualize and characterize biological processes. Among the imaging modalities, PET/SPECT and near-infrared (NIR) imaging provide synergistic properties that result in deep tissue penetration and up to cell-level resolution. Dual-modal PET/SPECT-NIR agents are commonly combined with a targeting ligand (e.g., antibody or small molecule) to engage biomolecules overexpressed in cancer, thereby enabling selective multimodal visualization of primary and metastatic tumors. The use of such agents for (i) preoperative patient selection and surgical planning and (ii) intraoperative FGS could improve surgical workflow and patient outcomes. However, the development of targeted dual-modal agents is a chemical challenge and a topic of ongoing research. In this review, we define key design considerations of targeted dual-modal imaging from a topological perspective, list targeted dual-modal probes disclosed in the last decade, review recent progress in the field of NIR fluorescent probe development, and highlight future directions in this rapidly developing field.

Establishment of Novel Neuroendocrine Carcinoma Patient-Derived Xenograft Models for Receptor Peptide-Targeted Therapy.

Gastroenteropancreatic neuroendocrine neoplasms (GEP NENs) are rare cancers consisting of neuroendocrine carcinomas (NECs) and neuroendocrine tumors (NETs), which have been increasing in incidence in recent years. Few cell lines and pre-clinical models exist for studying GEP NECs and NETs, limiting the ability to discover novel imaging and treatment modalities. To address this gap, we isolated tumor cells from cryopreserved patient GEP NECs and NETs and injected them into the flanks of immunocompromised mice to establish patient-derived xenograft (PDX) models. Two of six mice developed tumors (NEC913 and NEC1452). Over 80% of NEC913 and NEC1452 tumor cells stained positive for Ki67. NEC913 PDX tumors expressed neuroendocrine markers such as chromogranin A (CgA), synaptophysin (SYP), and somatostatin receptor-2 (SSTR2), whereas NEC1452 PDX tumors did not express SSTR2. Exome sequencing revealed loss of TP53 and RB1 in both NEC tumors. To demonstrate an application of these novel NEC PDX models for SSTR2-targeted peptide imaging, the NEC913 and NEC1452 cells were bilaterally injected into mice. Near infrared-labelled octreotide was administered and the fluorescent signal was specifically observed for the NEC913 SSTR2 positive tumors. These 2 GEP NEC PDX models serve as a valuable resource for GEP NEN therapy testing.

Characterization of Peptides Targeting Metastatic Tumor Cells as Probes for Cancer Detection and Vehicles for Therapy Delivery.

Metastasis is the leading cause of cancer-related deaths, and metastatic cancers remain largely incurable due to chemoresistance. Biomarkers of metastatic cells are lacking, and probes that could be used to detect and target metastases would be highly valuable. Here we hypothesize that metastatic cancer cells express cell-surface receptors that can be harnessed for identification of molecules homing to metastases. Screening a combinatorial library in a mouse mammary tumor model of spontaneous metastasis identified cyclic peptides with tropism for cancer cells disseminated to the lungs. Two lead peptides, CLRHSSKIC and CRAGVGRGC, bound murine and human cells derived from breast carcinoma and melanoma in culture and were selective for metastatic cells in vivo. In mice, peptide CRAGVGRGC radiolabeled with 67Ga for biodistribution analysis demonstrated selective probe homing to lung metastases. Moreover, systemic administration of 68Ga-labeled CRAGVGRGC enabled noninvasive imaging of lung metastases in mice by PET. A CRAGVGRGC-derived peptide induced apoptosis upon cell internalization in vitro and suppressed metastatic burden in vivo. Colocalization of CLRHSSKIC and CRAGVGRGC with N-cadherin+/E-cadherin- cells indicated that both peptides are selective for cancer cells that have undergone the epithelial-to-mesenchymal transition. We conclude that CRAGVGRGC is useful as a probe to facilitate the development of imaging modalities and therapies targeting metastases. SIGNIFICANCE: This study identifies new molecules that bind metastatic cells and demonstrates their application as noninvasive imaging probes and vehicles for cytotoxic therapy delivery in preclinical cancer models.

Receptor-Targeted Fluorescence-Guided Surgery With Low Molecular Weight Agents.

Cancer surgery remains the primary treatment option for most solid tumors and can be curative if all malignant cells are removed. Surgeons have historically relied on visual and tactile cues to maximize tumor resection, but clinical data suggest that relapse occurs partially due to incomplete cancer removal. As a result, the introduction of technologies that enhance the ability to visualize tumors in the operating room represents a pressing need. Such technologies have the potential to revolutionize the surgical standard-of-care by enabling real-time detection of surgical margins, subclinical residual disease, lymph node metastases and synchronous/metachronous tumors. Fluorescence-guided surgery (FGS) in the near-infrared (NIRF) spectrum has shown tremendous promise as an intraoperative imaging modality. An increasing number of clinical studies have demonstrated that tumor-selective FGS agents can improve the predictive value of fluorescence over non-targeted dyes. Whereas NIRF-labeled macromolecules (i.e., antibodies) spearheaded the widespread clinical translation of tumor-selective FGS drugs, peptides and small-molecules are emerging as valuable alternatives. Here, we first review the state-of-the-art of promising low molecular weight agents that are in clinical development for FGS; we then discuss the significance, application and constraints of emerging tumor-selective FGS technologies.

A proof-of-concept methodology to validate the in situ visualization of residual disease using cancer-targeted molecular agents in fluorescence-guided surgery.

INTRODUCTION: The clinical need for improved intraoperative tumor visualization has led to the development of targeted contrast agents for fluorescence-guided surgery (FGS). A key characteristic of these agents is their high tumor specificity, which could enable detection of residual lesions that would likely be missed by visual inspection. Here, we examine the utility of a promising somatostatin receptor subtype-2 (SSTR2)-targeted fluorescent agent for detecting residual disease in mouse xenografts using FGS and post-operative histopathological validation. METHODS: Mice (n=2) implanted with SSTR2 overexpressing tumors were injected with 2 nmol of the dual-labeled somatostatin analog, 67Ga-MMC(IR800)-TOC, and tumors were resected 48 h post-injection using traditional white light reflectance and palpation. Tumors underwent gamma counting and histopathology analysis. The wide-field FGS imaging platform (OnLume) was used to evaluate residual disease in situ under ambient light representative of an operating room. RESULTS: The tumor was resected with grossly negative margins using conventional inspection and palpation; however, additional in situ residual disease was found in the tumor cavity using FGS imaging. In situ fluorescent tumor contrast-to-noise ratios (CNRs) were 3.0 and 5.2. Agent accumulation was 7.72 and 8.20 %ID/g in tumors and 0.27 and 0.20 %ID/g in muscle. Fluorescence pixel values and gamma counts were highly correlated (r = 0.95, P < 0.048). H&E and IHC staining confirmed cancer positivity and SSTR2-overexpression, respectively. CONCLUSION: Our findings demonstrate that the use of clinically relevant fluorescence imaging instrumentation enhances the evaluation of promising FGS agents for in situ visualization of residual disease.

Development of a drug-device combination for fluorescence-guided surgery in neuroendocrine tumors.

SIGNIFICANCE: The use of cancer-targeted contrast agents in fluorescence-guided surgery (FGS) has the potential to improve intraoperative visualization of tumors and surgical margins. However, evaluation of their translational potential is challenging. AIM: We examined the utility of a somatostatin receptor subtype-2 (SSTR2)-targeted fluorescent agent in combination with a benchtop near-infrared fluorescence (NIRF) imaging system to visualize mouse xenografts under conditions that simulate the clinical FGS workflow for open surgical procedures. APPROACH: The dual-labeled somatostatin analog, Ga67-MMC(IR800)-TOC, was injected into mice (n = 24) implanted with SSTR2-expressing tumors and imaged with the customized OnLume NIRF imaging system (Madison, Wisconsin). In vivo and ex vivo imaging were performed under ambient light. The optimal dose (0.2, 0.5, and 2 nmol) and imaging time point (3, 24, 48, and 72 h) were determined using contrast-to-noise ratio (CNR) as the image quality parameter. Video captures of tumor resections were obtained to provide an FGS readout that is representative of clinical utility. Finally, a log-transformed linear regression model was fitted to assess congruence between fluorescence readouts and the underlying drug distribution. RESULTS: The drug-device combination provided high in vivo and ex vivo contrast (CNRs > 3, except lung at 3 h) at all time points with the optimal dose of 2 nmol. The optimal imaging time point was 24-h post-injection, where CNRs > 6.5 were achieved in tissues of interest (i.e., pancreas, small intestine, stomach, and lung). Intraoperative FGS showed excellent utility for examination of the tumor cavity pre- and post-resection. The relationship between fluorescence readouts and gamma counts was linear and strongly correlated (n = 334, R2 = 0.71; r = 0.84; P < 0.0001). CONCLUSION: The innovative OnLume NIRF imaging system enhanced the evaluation of Ga67-MMC(IR800)-TOC in tumor models. These components comprise a promising drug-device combination for FGS in patients with SSTR2-expressing tumors.

N,N-Dimethylsphingosine conjugates of poly-L-glutamic acid: synthesis, characterization, and initial biological evaluation.

Poly-L-glutamic acid (PGA) has previously been demonstrated to be an effective backbone for creating a hydrophilic prodrug of the established anti-tumor agent, paclitaxel, the active agent in Taxol; this approach has obviated the need for the toxic Cremophor excipient, used to enhance the solubility of paclitaxel in the clinical formulation. In order to form hydrophilic prodrugs of the hydrophobic pro-apoptotic sphingolipid, N,N-dimethylsphingosine (DMSP), PGA was condensed with DMSP, previously modified with coumarin to allow spectroscopic detection during conjugate synthesis, to yield PGA-DMSP. Conjugates with different loadings of DMSP were prepared and evaluated for in vitro cytotoxicity against two human breast adenocarcinoma cell lines. Time- and loading-dependent expression of cytotoxicity was observed, such that endpoints essentially equivalent to those observed with free-DMSP were achieved, but in a more protracted manner, consistent with prodrug behavior. PGA-DMSP was initially evaluated for toxicity in female nude mice, and administration of high net levels of DMSP, exceeding those achievable with free-DMSP, was well-tolerated. We propose that PGA-DMSP conjugates merit evaluation for anti-tumor efficacy in pre-clinical tumor models.

New Developments in Dual-Labeled Molecular Imaging Agents.

Intraoperative detection of tumors has had a profound impact on how cancer surgery is performed and addresses critical unmet needs in surgical oncology. Tumor deposits, margins, and residual cancer can be imaged through the use of fluorescent contrast agents during surgical procedures to complement visual and tactile guidance. The combination of fluorescent and nuclear contrast into a multimodality agent builds on these capabilities by adding quantitative, noninvasive nuclear imaging capabilities to intraoperative imaging. This review focuses on new strategies for the development and evaluation of targeted dual-labeled molecular imaging agents while highlighting the successful first-in-human application of this technique.

Specific Targeting of Somatostatin Receptor Subtype-2 for Fluorescence-Guided Surgery.

PURPOSE: Clinically available intraoperative imaging tools to assist surgeons in identifying occult lesions are limited and partially responsible for the high rate of disease recurrence in patients with neuroendocrine tumors (NET). Using the established clinical efficacy of radiolabeled somatostatin analogs as a model, we demonstrate the ability of a fluorescent somatostatin analog to selectively target tumors that overexpress somatostatin receptor subtype-2 (SSTR2) and demonstrate utility for fluorescence-guided surgery (FGS). EXPERIMENTAL DESIGN: A multimodality chelator (MMC) was used as a "radioactive linker" to synthesize the fluorescently labeled somatostatin analog, 67/68Ga-MMC(IR800)-TOC. In vivo studies were performed to determine the pharmacokinetic profile, optimal imaging time point, and specificity for SSTR2-expressing tissues. Meso- and microscopic imaging of resected tissues and frozen sections were also performed to further assess specific binding, and binding to human NETs was examined using surgical biospecimens from patients with pancreatic NETs. RESULTS: Direct labeling with 67Ga/68Ga provided quantitative biodistribution analysis that was in agreement with fluorescence data. Receptor-mediated uptake was observed in vivo and ex vivo at the macro-, meso-, and microscopic scales. Surgical biospecimens from patients with pancreatic NETs also displayed receptor-specific agent binding, allowing clear delineation of tumor boundaries that matched pathology findings. CONCLUSIONS: The radioactive utility of the MMC allowed us to validate the binding properties of a novel FGS agent that could have a broad impact on cancer outcomes by equipping surgeons with real-time intraoperative imaging capabilities.

A Modular Dual-Labeling Scaffold That Retains Agonistic Properties for Somatostatin Receptor Targeting.

Fluorescence-guided surgery is an emerging imaging technique that can enhance the ability of surgeons to detect tumors when compared with visual observation. To facilitate characterization, fluorescently labeled probes have been dual-labeled with a radionuclide to enable cross-validation with nuclear imaging. In this study, we selected the somatostatin receptor imaging agent DOTATOC as the foundation for developing a dual-labeled analog. We hypothesized that a customized dual-labeling approach with a multimodality chelation (MMC) scaffold would minimize steric effects of dye conjugation and retain agonist properties. Methods: An MMC conjugate (MMC-TOC) was synthesized on solid-phase and compared with an analog prepared using conventional methods (DA-TOC). Both analogs were conjugated to IRDye 800 using copper-free click chemistry. The resulting compounds, MMC(IR800)-TOC and DA(IR800)-TOC, were labeled with Cu and 64Cu and tested in vitro in somatostatin receptor subtype 2-overexpressing HEK-293 cells to assess agonist properties, and in AR42J rat pancreatic cancer cells to determine receptor binding characteristics. Multimodality imaging was performed in AR42J xenografts. Results: Cu-MMC(IR800)-TOC demonstrated higher potency for cyclic adenosine monophosphate inhibition (half maximal effective concentration [EC50]: 0.21 ± 0.18 vs. 1.38 ± 0.54 nM) and receptor internalization (EC50: 41.9 ± 29.8 vs. 455 ± 299 nM) than Cu-DA(IR800)-TOC. Radioactive uptake studies showed that blocking with octreotide caused a dose-dependent reduction in 64Cu-MMC(IR800)-TOC uptake whereas 64Cu-DA(IR800)-TOC was not affected. In vivo studies revealed higher tumor uptake for 64Cu-MMC(IR800)-TOC than 64Cu-DA(IR800)-TOC (5.2 ± 0.2 vs. 3.6 ± 0.4 percentage injected dose per gram). In vivo blocking studies with octreotide reduced tumor uptake of 64Cu-MMC(IR800)-TOC by 66%. Excretion of 64Cu-MMC(IR800)-TOC was primarily through the liver and spleen whereas 64Cu-DA(IR800)-TOC was cleared through the kidneys. Ex vivo analysis at 24 h confirmed PET/CT data by showing near-infrared fluorescence signal in tumors and a tumor-to-muscle ratio of 5.3 ± 0.8 as determined by γ-counting. Conclusion: The findings demonstrate that drug design affected receptor pharmacology and suggest that the MMC scaffold is a useful tool for the development of dual-labeled imaging agents.

Synthesis of a Fluorescently Labeled 68Ga-DOTA-TOC Analog for Somatostatin Receptor Targeting.

Fluorescently labeled imaging agents can identify surgical margins in real-time to help achieve complete resections and minimize the likelihood of local recurrence. However, photon attenuation limits fluorescence-based imaging to superficial lesions or lesions that are a few millimeters beneath the tissue surface. Contrast agents that are dual-labeled with a radionuclide and fluorescent dye can overcome this limitation and combine quantitative, whole-body nuclear imaging with intraoperative fluorescence imaging. Using a multimodality chelation (MMC) scaffold, IRDye 800CW was conjugated to the clinically used somatostatin analog, 68Ga-DOTA-TOC, to produce the dual-labeled analog, 68Ga-MMC(IRDye 800CW)-TOC, with high yield and specific activity. In vitro pharmacological assays demonstrated retention of receptor-targeting properties for the dual-labeled compound with robust internalization that was somatostatin receptor (SSTR) 2-mediated. Biodistribution studies in mice identified the kidneys as the primary excretion route for 68Ga-MMC(IRDye 800CW)-TOC, along with clearance via the reticuloendothelial system. Higher uptake was observed in most tissues compared to 68Ga-DOTA-TOC but decreased as a function of time. The combination of excellent specificity for SSTR2-expressing cells and suitable biodistribution indicate potential application of 68Ga-MMC(IRDye 800CW)-TOC for intraoperative detection of SSTR2-expressing tumors.

LGR5-Targeted Antibody-Drug Conjugate Eradicates Gastrointestinal Tumors and Prevents Recurrence.

Gastrointestinal cancer is one of the leading causes of cancer-related mortality in men and women worldwide. The adult stem cell marker LGR5 (leucine-rich repeat-containing, G protein-coupled receptor 5) is highly expressed in a significant fraction of gastrointestinal tumors of the colon, liver, pancreas, and stomach, relative to normal tissues. LGR5 is located on the cell surface and undergoes rapid, constitutive internalization independent of ligand. Furthermore, LGR5-high cancer cells have been shown to exhibit the properties of tumor-initiating cells or cancer stem cells (CSC). On the basis of these attributes, we generated two LGR5-targeting antibody-drug conjugates (ADC) by tethering the tubulin-inhibiting cytotoxic drug monomethyl auristatin E to a highly specific anti-LGR5 mAb via a protease cleavable or noncleavable chemical linker and compared them in receptor binding, cell internalization, and cytotoxic efficacy in cancer cells. Here, we show that both ADCs bind LGR5 with high specificity and equivalent nanomolar affinity and rapidly internalize to the lysosomes of LGR5-expressing gastrointestinal cancer cells. The anti-LGR5 ADCs effectively induced cytotoxicity in LGR5-high gastrointestinal cancer cells, but not in LGR5-negative or -knockdown cancer cell lines. Overall, we demonstrate that the cleavable ADC exhibited higher potency in vitro and was able to eradicate tumors and prevent recurrence in a xenograft model of colon cancer. These findings provide preclinical evidence for the potential of LGR5-targeting ADCs as effective new therapeutics for the treatment and eradication of gastrointestinal tumors and CSCs with high LGR5 expression. Mol Cancer Ther; 15(7); 1580-90. ©2016 AACR.

Molecular inhibition of prostaglandin E2 with GW627368X: Therapeutic potential and preclinical safety assessment in mouse sarcoma model.

Prostaglandin E2, the major COX-2 product, acts via 4 functionally distinct prostanoid receptors, EP(1-4). PGE-2, through its receptors, feeds back to positively increase COX-2 expression augmenting its own synthesis thereby driving angiogenesis, while suppressing apoptosis and innate immunity. In addition to the well characterized PGE2/EP4/cAMP/PKA/CREB, EP4 activation increases GSK3 phosphorylation via PI3K and Akt consequently reducing ß-catenin phosphorylation. EP4 induces angiogenesis by enhancing VEGF production via ERK activation. These effects of EP4 are asserted either directly or via EGFR transactivation depending on the type of cancer. In view of the safety concerns regarding long term use of COX-2 inhibitors and to find more effective alternatives, we evaluated the potential of EP4 prostanoid receptor as a target for treating cancer progression using a highly selective EP4 antagonist, 4-(4,9-diethoxy-1,3-dihydro-1-oxo-2H-benz[f]isoindol-2-yl)-N-(phenylsulfonyl)-benzeneacetamide. Oral administration of GW627368X showed significant tumor regression characterized by tumor reduction and induction of apoptosis. Reduction in prostaglandin E2 synthesis also led to reduced level of VEGF in plasma. Regulation of multiple pathways downstream of EP4 was evident by down regulation of COX-2, p-Akt, p-MAPK and p-EGFR. Considering wide distribution of the EP4 prostanoid receptor in major organs and the array of physiological processes it contributes to, the safety profile of the drug was analyzed. No major organ toxicity, immunosupression, behavioral change or change in blood parameters attributable to the drug was observed. The results assert the significance of EP4 prostanoid receptor as a therapeutic target as well as the safety of EP4 blockade by GW627368X.