Fluorescence Imaging of the Ureter in Minimally Invasive Pelvic Surgery.

STUDY OBJECTIVE: Determine near-optimal dose, safety, and efficacy of nerindocianine in pelvic ureter detection with near-infrared fluorescence imaging in women undergoing minimally invasive pelvic surgery with 3 Food and Drug Administration-cleared imaging systems. DESIGN: Open label, phase 1/2a study. SETTING: University of Alabama at Birmingham. PATIENTS: Forty-one female subjects undergoing minimally invasive gynecologic surgery. INTERVENTIONS: Subjects received a single dose of nerindocianine sodium, starting at 0.06-mg/kg body weight and increased/decreased until the near-optimal dose was determined (part A). Examine the degree of concordance between endoscopic and robotic devices (part B). MEASUREMENTS AND MAIN RESULTS: In part A, composite scores were collected every 10 minutes for 30 minutes and then every 15 minutes through 90 minutes using a scale measuring the anatomy/laterality of ureter visualization. In part B (paired imaging system efficacy), 2 cohorts of 8 subjects each received the near-optimal dose. Composite scores for visualization of the ureter were collected at 10 and 30 minutes postinfusion with the Firefly Imaging System and either the PINPOINT or 1588 AIM endoscope. Composite scores were compared to examine the degree of concordance between devices. Part A comprised 25 total subjects enrolled in dosing groups 1, 2, and 3 (0.06-, 0.12-, and 0.045-mg/kg, respectively). Median time to first ureter visualization was 10 minutes (all groups). The nerindocianine 0.06-mg/kg and 0.12-mg/kg groups had longer length of time of visualization than the 0.045-mg/kg group, resulting in the selection of 0.06 mg/kg as the near-optimal dose. Part B enrolled 16 total subjects in 2 groups dosed at 0.06 mg/kg. Efficacy analysis showed no statistically significant difference in composite scores with Firefly versus PINPOINT or 1588 AIM. CONCLUSION: Nerindocianine was well tolerated with visualization of the ureter demonstrated in 88.9% of the subjects through 90 minutes postdosing. No meaningful visualization differences were observed among the Food and Drug Administration-cleared surgical imaging systems used.

SPARC-Independent Delivery of Nab-Paclitaxel without Depleting Tumor Stroma in Patient-Derived Pancreatic Cancer Xenografts.

The study goal was to examine the relationship between nab-paclitaxel delivery and SPARC (secreted protein acidic and rich in cysteine) expression in pancreatic tumor xenografts and to determine the antistromal effect of nab-paclitaxel, which may affect tumor vascular perfusion. SPARC-positive and -negative mice bearing Panc02 tumor xenografts (n = 5-6/group) were injected with IRDye 800CW (IR800)-labeled nab-paclitaxel. After 24 hours, tumors were collected and stained with DL650-labeled anti-SPARC antibody, and the correlation between nab-paclitaxel and SPARC distributions was examined. Eight groups of mice bearing either Panc039 or Panc198 patient-derived xenografts (PDX; 4 groups/model, 5 animals/group) were untreated (served as control) or treated with gemcitabine (100 mg/kg body weight, i.p., twice per week), nab-paclitaxel (30 mg/kg body weight, i.v., for 5 consecutive days), and these agents in combination, respectively, for 3 weeks, and tumor volume and perfusion changes were assessed using T2-weighted MRI and dynamic contrast-enhanced (DCE) MRI, respectively. All tumors were collected and stained with Masson's Trichrome Stain, followed by a blinded comparative analysis of tumor stroma density. IR800-nab-paclitaxel was mainly distributed in tumor stromal tissue, but nab-paclitaxel and SPARC distributions were minimally correlated in either SPARC-positive or -negative animals. Nab-paclitaxel treatment neither decreased tumor stroma nor increased tumor vascular perfusion in either PDX model when compared with control groups. These data suggest that the specific tumor delivery of nab-paclitaxel is not directly related to SPARC expression, and nab-paclitaxel does not deplete tumor stroma in general. Mol Cancer Ther; 15(4); 680-8. ©2016 AACR.

Fluorescence-guided resection of experimental malignant glioma using cetuximab-IRDye 800CW.

The standard treatment for glioblastoma multiforme (GBM) remains maximal safe surgical resection. Here, we evaluated the ability of a systemically administered antibody-dye probe conjugate (cetuximab-IRDye 800CW) to provide sufficient fluorescent contrast for surgical resection of disease in both subcutaneous and orthotopic animal models of GBM. Multiple luciferase-positive GBM cell lines (D-54MG, U-87MG, and U-251MG; n = 5) were implanted in mouse flank and tumors were fluorescently imaged daily using a closed-field near-infrared (NIR) system after cetuximab-IRDye 800CW systemic administration. Orthotopic models were also generated (n = 5), and tumor resection was performed under white light and fluorescence guidance using an FDA-approved wide-field NIR imaging system. Residual tumor was monitored using luciferase imaging. Immunohistochemistry was performed to characterize tumor fluorescence, epidermal growth factor receptor (EGFR) expression, and vessel density. Daily imaging of tumors revealed an average tumor-to-background (TBR) of 4.5 for U-87MG, 4.1 for D-54MG, and 3.7 for U-251MG. Fluorescence intensity within the tumors peaked on day-1 after cetuximab-IRDye 800CW administration, however the TBR increased over time in two of the three cell lines. For the orthotopic model, TBR on surgery day ranged from 19 to 23 during wide-field, intraoperative imaging. Surgical resection under white light on day 3 after cetuximab-IRDye 800CW resulted in an average 41% reduction in luciferase signal while fluorescence-guided resection using wide-field NIR imaging resulted in a significantly (P = 0.001) greater reduction in luciferase signal (87%). Reduction of luciferase signal was found to correlate (R (2) = 0.99) with reduction in fluorescence intensity. Fluorescence intensity was found to correlate (P < 0.05) with EGFR expression in D-54MG and U-251MG tumor types but not U-87MG. However, tumor fluorescence was found to correlate with vessel density for the U-87MG tumors. Here we show systemic administration of cetuximab-IRDye 800CW in combination with wide-field NIR imaging provided robust and specific fluorescence contrast for successful localization of disease in subcutaneous and orthotopic animal models of GBM.

Pharmacokinetic and Biodistribution Assessment of a Near Infrared-Labeled PSMA-Specific Small Molecule in Tumor-Bearing Mice.

Prostate cancer is the most frequently diagnosed cancer in men and often requires surgery. Use of near infrared (NIR) technologies to perform image-guided surgery may improve accurate delineation of tumor margins. To facilitate preclinical testing of such outcomes, here we developed and characterized a PSMA-targeted small molecule, YC-27. IRDye 800CW was conjugated to YC-27 or an anti-PSMA antibody used for reference. Human 22Rv1, PC3M-LN4, and/or LNCaP prostate tumor cells were exposed to the labeled compounds. In vivo targeting and clearance properties were determined in tumor-bearing mice. Organs and tumors were excised and imaged to assess probe localization. YC-27 exhibited a dose dependent increase in signal upon binding. Binding specificity and internalization were visualized by microscopy. In vitro and in vivo blocking studies confirmed YC-27 specificity. In vivo, YC-27 showed good tumor delineation and tissue contrast at doses as low as 0.25 nmole. YC-27 was cleared via the kidneys but bound the proximal tubules of the renal cortex and epididymis. Since PSMA is also broadly expressed on the neovasculature of most tumors, we expect YC-27 will have clinical utility for image-guided surgery and tumor resections.

A comparative study of affibody, panitumumab, and EGF for near-infrared fluorescence imaging of EGFR- and EGFRvIII-expressing tumors.

Aberrant overexpression and/or activation of epidermal growth factor receptor (EGFR) is associated with many types of cancers. EGFR variant III (EGFRvIII) is a common in-frame deletion mutant, which lacks a large part of the extracellular portion (exons 2-7), including components of the ligand-binding domain. Although EGFR has been extensively studied as a molecular imaging target, information about EGFRvIII-targeted molecular imaging is lacking. In this study, the EGFR-specific affibody, therapeutic antibody panitumumab, and ligand EGF were labeled with IRDye 800CW (Ex/Em: 774/789 nm), yielding Aff800, Pan800, and EGF800, respectively. The binding affinities of the labeled agents were compared in cell-based assays using a rat glioma cell line F98 parental (F98-p) lacking EGFR expression, and 2 F98-derived transgenic cell lines expressing EGFR or EGFRvIII (designated as F98-EGFR and F98-vIII, respectively). Results showed that all agents could bind to F98-EGFR, with Pan800 having the highest binding affinity, followed by Aff800 and EGF800. Pan800 and Aff800, but not EGF800, also bound to F98-vIII. In vivo animal imaging demonstrated that compared with F98-p tumors, F98-EGFR tumors generated higher signals with all three agents. However, in the case of F98-vIII, only Pan800 and Aff800 signals were higher. Analysis of tissue lysates showed that a large portion of Pan800 was degraded into small fragments in F98-EGFR and F98-vIII tumors, possibly due to proteolytic digestion after its specific binding and internalization. In conclusion, Pan800 and Aff800 could be used as imaging agents for both wild-type EGFR and EGFRvIII, whereas EGF800 only targets wild-type EGFR.

Use of monoclonal antibody-IRDye800CW bioconjugates in the resection of breast cancer.

BACKGROUND: Complete surgical resection of breast cancer is a powerful determinant of patient outcome, and failure to achieve negative margins results in reoperation in between 30% and 60% of patients. We hypothesize that repurposing Food and Drug Administration-approved antibodies as tumor-targeting diagnostic molecules can function as optical contrast agents to identify the boundaries of malignant tissue intraoperatively. MATERIALS AND METHODS: The monoclonal antibodies bevacizumab, cetuximab, panitumumab, trastuzumab, and tocilizumab were covalently linked to a near-infrared fluorescence probe (IRDye800CW) and in vitro binding assays were performed to confirm ligand-specific binding. Nude mice bearing human breast cancer flank tumors were intravenously injected with the antibody-IRDye800 bioconjugates and imaged over time. Tumor resections were performed using the SPY and Pearl Impulse systems, and the presence or absence of tumor was confirmed by conventional and fluorescence histology. RESULTS: Tumor was distinguishable from normal tissue using both SPY and Pearl systems, with both platforms being able to detect tumor as small as 0.5 mg. Serial surgical resections demonstrated that real-time fluorescence can differentiate subclinical segments of disease. Pathologic examination of samples by conventional and optical histology using the Odyssey scanner confirmed that the bioconjugates were specific for tumor cells and allowed accurate differentiation of malignant areas from normal tissue. CONCLUSIONS: Human breast cancer tumors can be imaged in vivo with multiple optical imaging platforms using near-infrared fluorescently labeled antibodies. These data support additional preclinical investigations for improving the surgical resection of malignancies with the goal of eventual clinical translation.

Near-infrared optical imaging for monitoring the regeneration of osteogenic tissue-engineered constructs.

Millions of cases of bone injury or loss due to trauma, osteoporosis, and cancer occur in the United States each year. Because bone is limited in its ability to regenerate, alternative therapy approaches are needed. Bone tissue engineering has the potential to correct musculoskeletal disorders through the development of cell-based substitutes for osteogenic tissue replacement. Multiple medical imaging techniques such as magnetic resonance microscopy (MRM) were investigated recently; these techniques are able to provide useful information on the anatomical and structural changes of developing bone. However, there is a need for noninvasive approaches to evaluate biochemical constituents and consequent compositional development associated with growing osteogenic constructs. In this study, near-infrared (NIR) optical imaging with a bone-specific NIR-targeted probe, IRDye(®) 800CW BoneTag™ (800CW BT), was applied in this study to longitudinally visualize regions of mineralization of tissue-engineered bone constructs in vivo. A fluorescent cell-based assay was performed to confirm the preferential binding of 800CW BT to the mineralized matrix of differentiated osteogenically driven human mesenchymal stem cells (hMSCs) in vitro. The hMSCs were seeded onto a biocompatible gelatin scaffold, allowed to develop, and implanted into a mouse model. Engineered constructs were examined in vivo using NIR imaging for bone mineralization, paired with MRM for verification of developing tissue. Results showed that NIR imaging with 800CW BT labeling can effectively assess the calcification of the developing osteogenic constructs, which is consistent with the analysis of excised tissue using NIR microscopy and histology. In conclusion, this study evaluated bone-like function of regenerating bone through tracking calcium deposition via NIR optical imaging with a fluorophore-labeled probe in a noninvasive manner.

Characterization of IRDye 800CW chlorotoxin as a targeting agent for brain tumors.

Primary brain tumors present significant challenges for surgical resection because of their location and the frequent occurrence of malignant projections extending beyond the primary tumor. Visualization of the tumor margins during surgery is critical for a favorable outcome. We report the use of IRDye 800CW chlorotoxin (CLTX) as a targeted imaging agent for brain tumors in a spontaneous mouse model of medulloblastoma, ND2:SmoA1. Specificity and functionality of the targeted agent were confirmed in cell-based assays. Tumors were detected by magnetic resonance imaging and IRDye 800CW CLTX administered to individual animals for optical imaging at 1-month increments. The integrity of the blood-brain barrier (BBB) was measured by Evan's Blue perfusion prior to sacrifice. Results show that IRDye 800CW CLTX specifically targeted tumor tissue. The extravasation of Evan's Blue was observed in all tumors, suggesting that the presence of the tumors can introduce alterations in the permeability of the BBB. Because increased vascular permeability was observed early in the disease model, larger dye-labeled imaging agents that exceed current BBB size restrictions may warrant renewed consideration as candidates for tumor detection and surgical resection. Our study provides data characterizing in vitro and in vivo use of IRDye 800CW CLTX as a broadly applicable tumor imaging agent.

Fluorescently labeled therapeutic antibodies for detection of microscopic melanoma.

OBJECTIVES/HYPOTHESIS: Detection of microscopic disease during surgical resection of melanoma remains a significant challenge. To assess real-time optical imaging for visualization of microscopic cancer, we evaluated three US Food and Drug Administration (FDA)-approved therapeutic monoclonal antibodies. STUDY DESIGN: Prospective, basic science. METHODS: Melanoma cell lines (A375 and SKMEL5) were xenografted into the ears of immunodeficient mice. Bevacizumab, panitumumab, tocilizumab, or a nonspecific immunoglobin G (IgG) were covalently linked to a near-infrared (NIR) fluorescent probe (IRDye800CW) and systemically injected. Primary tumors were imaged and then resected under fluorescent guidance using the SPY (Novadaq, Toronto, Ontario, Canada), an NIR imaging system used in plastic and reconstructive surgeries to evaluate perfusion. Mice were also imaged with the Pearl Impulse small animal imager (LI-COR Biosciences, Lincoln, NE), an NIR imaging system designed for use with IRDye800CW. Postresection, small tissue fragments were fluorescently imaged and the presence of tumor subsequently confirmed by correlation with histology. RESULTS: All fluorescently labeled therapeutic monoclonal antibodies could adequately delineate tumor from normal tissue based on tumor-to-background ratios (TBR) compared to IgG-IRDye800CW. On serial imaging, panitumumab achieved the highest TBRs with both SPY and Pearl (3.8 and 6.6, respectively). When used to guide resections, the antibody-dye conjugates generated TBRs in the range of 1.3 to 2.2 (average, 1.6) using the SPY and 1.9 to 6.3 (average, 2.7) using the Pearl. There was no significant difference among the antibodies with either imaging modality or cell line (one-way analysis of variance). CONCLUSIONS: Our data suggest that FDA-approved antibodies may be suitable targeting agents for the intraoperative fluorescent detection of melanoma.

Integrin αvß3-targeted IRDye 800CW near-infrared imaging of glioblastoma.

PURPOSE: Integrin α(v)ß(3) plays an important role in tumor angiogenesis, growth, and metastasis. We have tested a targeted probe to visualize integrin receptor expression in glioblastomas using near-infrared fluorescent (NIRF) imaging. EXPERIMENTAL DESIGN: A transgenic glioblastoma mouse model (RCAS-PDGF-driven/tv-a glioblastoma, which mimics the infiltrative growth pattern of human glioblastomas) and two human orthotopic glioblastoma models (U-87 MG with high integrin ß(3) expression and TS543 with low integrin ß(3) expression) were studied. An integrin-targeting NIRF probe, IRDye 800CW-cyclic-RGD peptide (IRDye 800CW-RGD), was tested by in vivo and ex vivo NIRF imaging. RESULTS: We show that the IRDye 800CW-RGD peptide: (i) specifically binds to integrin receptors; (ii) is selectively localized to glioblastoma tissue with overexpressed integrin receptors and is retained over prolonged periods of time; (iii) is associated with minimal autofluorescence and photobleaching because of imaging at 800 nm; (iv) provides delineation of tumor tissue with high precision because of a high tumor-to-normal brain fluorescence ratio (79.7 ± 6.9, 31.2 ± 2.8, and 16.3 ± 1.3) in the U-87 MG, RCAS-PDGF, and TS543 models, respectively; P < 0.01); and (v) enables fluorescence-guided glioblastoma resection. Importantly, small foci of residual fluorescence were observed after resection was completed using white light imaging alone, and these fluorescent foci were shown to represent residual tumor tissue by histology. CONCLUSIONS: NIRF imaging with the IRDye 800CW-RGD probe provides a simple, rapid, low-cost, nonradioactive, and highly translatable approach for improved intraoperative glioblastoma visualization and resection. It also has the potential to serve as an imaging platform for noninvasive cancer detection and drug efficacy evaluation studies.

Near-infrared fluorescence imaging of mammalian cells and xenograft tumors with SNAP-tag.

Fluorescence in the near-infrared (NIR) spectral region is suitable for in vivo imaging due to its reduced background and high penetration capability compared to visible fluorescence. SNAP(f) is a fast-labeling variant of SNAP-tag that reacts with a fluorescent dye-conjugated benzylguanine (BG) substrate, leading to covalent attachment of the fluorescent dye to the SNAP(f). This property makes SNAP(f) a valuable tool for fluorescence imaging. The NIR fluorescent substrate BG-800, a conjugate between BG and IRDye 800CW, was synthesized and characterized in this study. HEK293, MDA-MB-231 and SK-OV-3 cells stably expressing SNAP(f)-Beta-2 adrenergic receptor (SNAP(f)-ADRß2) fusion protein were created. The ADRß2 portion of the protein directs the localization of the protein to the cell membrane. The expression of SNAP(f)-ADRß2 in the stable cell lines was confirmed by the reaction between BG-800 substrate and cell lysates. Microscopic examination confirmed that SNAP(f)-ADRß2 was localized on the cell membrane. The signal intensity of the labeled cells was dependent on the BG-800 concentration. In vivo imaging study showed that BG-800 could be used to visualize xenograph tumors expressing SNAP(f)-ADRß2. However, the background signal was relatively high, which may be a reflection of non-specific accumulation of BG-800 in the skin. To address the background issue, quenched substrates that only fluoresce upon reaction with SNAP-tag were synthesized and characterized. Although the fluorescence was successfully quenched, in vivo imaging with the quenched substrate CBG-800-PEG-QC1 failed to visualize the SNAP(f)-ADRß2 expressing tumor, possibly due to the reduced reaction rate. Further improvement is needed to apply this system for in vivo imaging.

Near-infrared-labeled tetracycline derivative is an effective marker of bone deposition in mice.

Bone-specific compounds have been used effectively for the detection of bone mineralization, growth, and morphological changes. These agents typically contain iminodiacetic acid groups that can form complexes with apatite and fluoresce in the visible spectrum. We exploited a subset of these chemical chelators to produce a near-infrared (NIR) optical bone marker for preclinical animal imaging. By conjugating target compounds to IRDye 800CW, we extended the effective fluorescence signal detection to the NIR region without affecting the compound's ability to function as a marker of the mineralization process. Calcein and a tetracycline derivative (BoneTag agent [BT]) bound specifically to differentiated mineralized osteoblast cultures, with the latter exhibiting 6-fold higher signal intensities. Subsequent in vivo testing demonstrated effective skeletal labeling with IRDye 800CW BT. We were able to identify a changing mineralization front in bone sections from (i) normal growing mice injected with IRDye 800CW BT 6weeks prior to the administration of IRDye 680 BT and (ii) an osteoporosis mouse model comparing cortical bone in sham-treated and ovariectomized mice. These results provide evidence that the NIR-labeled BT is effective as a general marker of skeletal features and an indicator of the bone mineralization and remodeling processes.

In vivo imaging of xenograft tumors using an epidermal growth factor receptor-specific affibody molecule labeled with a near-infrared fluorophore.

Overexpression of epidermal growth factor receptor (EGFR) is associated with many types of cancers. It is of great interest to noninvasively image the EGFR expression in vivo. In this study, we labeled an EGFR-specific Affibody molecule (Eaff) with a near-infrared (NIR) dye IRDye800CW maleimide and tested the binding of this labeled molecule (Eaff800) in cell culture and xenograft mouse tumor models. Unlike EGF, Eaff did not activate the EGFR signaling pathway. Results showed that Eaff800 was bound and taken up specifically by EGFR-overexpressing A431 cells. When Eaff800 was intravenously injected into nude mice bearing A431 xenograft tumors, the tumor could be identified 1 hour after injection and it became most prominent after 1 day. Images of dissected tissue sections demonstrated that the accumulation of Eaff800 was highest in the liver, followed by the tumor and kidney. Moreover, in combination with a human EGFR type 2 (HER2)-specific probe Haff682, Eaff800 could be used to distinguish between EGFR- and HER2-overexpressing tumors. Interestingly, the organ distribution pattern and the clearance rate of Eaff800 were different from those of Haff682. In conclusion, Eaff molecule labeled with a NIR fluorophore is a promising molecular imaging agent for EGFR-overexpressing tumors.

Spontaneous metastasis of prostate cancer is promoted by excess hyaluronan synthesis and processing.

Accumulation of extracellular hyaluronan (HA) and its processing enzyme, the hyaluronidase Hyal1, predicts invasive, metastatic progression of human prostate cancer. To dissect the roles of hyaluronan synthases (HAS) and Hyal1 in tumorigenesis and metastasis, we selected nonmetastatic 22Rv1 prostate tumor cells that overexpress HAS2, HAS3, or Hyal1 individually, and compared these cells with co-transfectants expressing Hyal1 + HAS2 or Hyal1 + HAS3. Cells expressing only HAS were less tumorigenic than vector control transfectants on orthotopic injection into mice. In contrast, cells co-expressing Hyal1 + HAS2 or Hyal1 + HAS3 showed greater than sixfold and twofold increases in tumorigenesis, respectively. Fluorescence and histological quantification revealed spontaneous lymph node metastasis in all Hyal1 transfectant-implanted mice, and node burden increased an additional twofold when Hyal1 and HAS were co-expressed. Cells only expressing HAS were not metastatic. Thus, excess HA synthesis and processing in concert accelerate the acquisition of a metastatic phenotype by prostate tumor cells. Intratumoral vascularity did not correlate with either tumor size or metastatic potential. Analysis of cell cycle progression revealed shortened doubling times of Hyal1-expressing cells. Both adhesion and motility on extracellular matrix were diminished in HA-overproducing cells; however, motility was increased twofold by Hyal1 expression and fourfold to sixfold by Hyal1/HAS co-expression, in close agreement with observed metastatic potential. This is the first comprehensive examination of these enzymes in a relevant prostate cancer microenvironment.

Characterization and performance of a near-infrared 2-deoxyglucose optical imaging agent for mouse cancer models.

Malignant neoplasms exhibit an elevated rate of glycolysis over normal cells. This characteristic can be exploited for optical imaging of tumors in mice. A near-infrared fluorophore, IRDye 800CW, emission maximum 794 nm, was conjugated to 2-deoxyglucose (2-DG). An immunofluorescent cell-based assay was used to evaluate specificity and sensitivity of the conjugate in cultured cell monolayers. Dose-dependent uptake was established with increasing concentrations of IRDye 800CW 2-DG for epithelial and prostate carcinomas. IRDye 800CW 2-DG was specifically blocked by an antibody against GLUT1 glucose transporter, and by excess unlabeled 2-DG or d-glucose. Signal was increased by a phorbol ester activator of glucose transport. Fluorescence microscopy data confirmed localization of the conjugate in the cytoplasm. Subsequent in vivo studies optimized dose, clearance, and timing for signal capture in nude mouse xenografts. In all cases, tumors were clearly imaged with good signal-to-noise characteristics. These data indicate that IRDye 800CW 2-DG is a broadly applicable optical imaging agent for in vivo imaging of neoplasms in mice.

beta-Galactosidase activity assay using far-red-shifted fluorescent substrate DDAOG.

beta-Galactosidase (beta-gal) is commonly used as a reporter gene in biological research, and a wide variety of substrates have been developed to assay its activity. One substrate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) beta-d-galactopyranoside (DDAOG), can be cleaved by beta-gal to produce 7-hydroxy-9H(I,3-dichloro-9,9-dimethylacridin-2-one) (DDAO). On excitation, DDAO generates a far-red-shifted fluorescent signal. Using this substrate, we developed a beta-gal activity assay method. The DDAO signal was stable for at least 18h. The signal intensity was linearly related to both the enzyme amount and substrate concentration. An optimized buffer for the beta-gal/DDAOG assay was also formulated. When compared with the colorimetric substrate o-nitrophenyl-beta-d-galactopyranoside (ONPG), the signal-to-background ratio of the DDAOG method was approximately 12-fold higher. The beta-gal/DDAOG assay method was also tested in transiently transfected cells employing both pharmacologically and genetically inducible gene expression systems. The ability to detect signal induction is comparable to a similar assay using luciferase as the signal generating moiety. The beta-gal/DDAOG assay method should provide a fluorescent reporter assay system for the wide variety of beta-gal systems currently in use.

Purification method directly influences effectiveness of an epidermal growth factor-coupled targeting agent for noninvasive tumor detection in mice.

Receptor targeting is an effective method of enhancing fluorescence signal in tumors for optical imaging. We previously used epidermal growth factor (EGF) conjugated to IRDye 800CW to detect and track orthotopic prostate tumors in mice. In this study, our goal was to identify a reliable assay for targeting agent integrity in vitro that correlated with signal strength in vivo. Binding of IRDye 800CW EGF to intact A431 human epidermoid carcinoma cells was quantified in a microplate assay. Specificity was confirmed by competition with unlabeled EGF or monoclonal antibody blocking. Biological activity of intact and damaged targeting agents relative to unlabeled EGF was determined by binding and stimulation of extracellular signal-regulated kinase (ERK) phosphorylation. Both assays indicated a reduction of up to 60% of the fluorescence intensity with damaged agents. Using a research prototype imaging system optimized for IRDye 800CW detection, we compared the efficacy of intact and damaged targeting agents for imaging subcutaneous tumors in mice. In live animal images and in sections of the excised tumors, damaged targeting agents consistently yielded diminished fluorescence signals corresponding to the reduction observed in microplate assays. This is the first study to directly correlate targeting agent signal strength in whole cell binding, In-Cell Western, and in vivo near-infrared imaging.

Hyaluronidase expression induces prostate tumor metastasis in an orthotopic mouse model.

Molecular mechanisms of prostate cancer progression are frequently studied in mice by orthotopic injection of aggressive cell lines, which yield primary tumors that spontaneously metastasize to lymph nodes. In this report, we characterized the human prostate carcinoma cell line 22Rv1 in an orthotopic system and evaluated the functional relevance of the hyaluronidase Hyal1, a correlate of invasive human prostate cancer, to progression in this model. To provide real-time insights into these processes, we first validated use of an epidermal growth factor-conjugated fluorophore to illuminate orthotopic prostate tumors and their metastases in whole animal imaging. Animals receiving intraprostatic injections were tracked throughout a 6-week period. Tumor sizes were correlated 92% with total fluorescence intensities of 22 prostate tumors. In contrast to the highly tumorigenic and metastatic PC3M-LN4 cells, the 22Rv1 line was orthotopically tumorigenic but not metastatic, despite larger tumor sizes. Lymph node metastasis was successfully imaged in animals with PC3M-LN4 tumors on endpoint dissection. Stable transfection of 22Rv1 cells with Hyal1 did not alter growth kinetics of primary orthotopic tumors, but all animals implanted with Hyal1 transfectants exhibited tumor-positive para-aortic lymph nodes. Hyal1 is implicated as an inducer of prostate cancer metastatic progression.

A cell-based immunocytochemical assay for monitoring kinase signaling pathways and drug efficacy.

Protein kinases play important roles in many disease processes and are primary targets for drug development. Because cellular phosphorylation cascades are complex multidirectional pathways, the behavior of a drug in a biochemical enzyme assay may not accurately reflect its performance in the context of a whole cell. We have developed a near-infrared cytoblot assay that can be used to investigate both kinase signaling and effects of kinase inhibitors. Adherent cells were grown in either 96- or 384-well plates. Following stimulation, protein phosphorylation was detected immunohistochemically by simultaneous staining with two primary antibodies: a phospho-specific primary and normalization antibody that recognized either the target protein regardless of phosphorylation status (pan protein) or a housekeeping protein. Secondary antibodies labeled with two spectrally distinct near-infrared dyes were used for visualization. Nuclear staining with TO-PRO-3 was also used in place of the normalization antibody. Normalization for well-to-well variability was accomplished by ratiometric analysis of the two wavelengths. The near-infrared cytoblot was used to analyze phosphorylation of EGFR, Akt, Stat3, MEK 1, and ERK1/2. This assay format was also able to simultaneously assess the phosphorylation of multiple signaling proteins in response to known kinase inhibitors. We observed that the IC50 for the EGFR inhibitor PD168393 was similar for EGFR and Stat3 but was significantly higher for ERK1/2, a downstream modulator of EGFR function. The observation that the receptor and its effectors show different IC50 values for the same inhibitory drug could be important for target selection in drug development.