Dual probe with fluorescent and magnetic properties for imaging solid tumor xenografts

A dual probe with fluorescent and magnetic reporter groups was constructed by linkage of the near-infrared (NIR) fluorescent transferrin conjugate (Tf(NIR)) on the surface of contrast agent-encapsulated cationic liposome (Lip-CA). This probe was used for magnetic resonance imaging (MRI) and optical imaging of MDA-MB-231-luc breast cancer cells grown as a monolayer in vitro and as solid tumor xenografts in nude mice. Confocal microscopy, optical imaging, and MRI showed a dramatic increase of in vitro cellular uptake of the fluorescent and magnetic reporter groups from the probe compared with the uptake of contrast agent or Lip-CA alone. Pretreatment with transferrin (Tf) blocked uptake of the probe reporters, indicating the importance and specificity of the Tf moiety for targeting. Intravenous administration of the dual probe to nude mice significantly enhanced the tumor contrast in MRI, and preferential accumulation of the fluorescent signal was clearly seen in NIR-based optical images. More interestingly, the contrast enhancement in MRI showed a heterogeneous pattern within tumors, which reflected the tumor's morphologic heterogeneity. These results indicate that the newly developed dual probe enhances the tumor image contrast and is superior to contrast agent alone for identifying the tumor pathologic features on the basis of MRI but also is suitable for NIR-based optical imaging.     

In vivo method to monitor changes in HER2 expression using near-infrared fluorescence imaging

Human epidermal growth factor receptor type 2 (HER2) is a well-known biomarker that is overexpressed in many breast carcinomas. HER2 expression level is an important factor to optimize the therapeutic strategy and monitor the treatment. We used albumin binding domain-fused HER2-specific Affibody molecules, labeled with Alexa Fluor750 dye, to characterize HER2 expression in vivo. Near-infrared optical imaging studies were carried out using mice with subcutaneous HER2-positive tumors. Animals were divided into groups of five: no treatment and 12 hours and 1 week after treatment of the tumors with the Hsp90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG). The compartmental ligands-receptor model, describing binding kinetics, was used to evaluate HER2 expression from the time sequence of the fluorescence images after the intravenous probe injection. The normalized rate of accumulation of the specific fluorescent biomarkers, estimated from this time sequence, linearly correlates with the conventional ex vivo enzyme-linked immunosorbent assay (ELISA) readings for the same tumor. Such correspondence makes properly arranged fluorescence imaging an excellent candidate for estimating HER2 overexpression in tumors, complementing ELISA and other ex vivo assays. Application of this method to the fluorescence data from HER2-positive xenografts reveals that the 17-DMAG treatment results in downregulation of HER2. Application of the AngioSense 750 probe confirmed the antiangiogenic effect of 17-DMAG found with Affibody-Alexa Fluor 750 conjugate.     

In vitro and in vivo evaluation of Alexa Fluor 680-bombesin[7-14]NH2 peptide conjugate, a high-affinity fluorescent probe with high selectivity for the gastrin-releasing peptide receptor

Gastrin-releasing peptide (GRP) receptors are overexpressed on several types of human cancer cells, including breast, prostate, small cell lung, and pancreatic cancers. Bombesin (BBN), a 14-amino acid peptide that is an analogue of human GRP, binds to GRP receptors with very high affinity and specificity. The aim of this study was to develop a new fluorescent probe based on BBN having high tumor uptake and optimal pharmacokinetics for specific targeting and optical imaging of human breast cancer tissue. In this study, solid-phase peptide synthesis was used to produce H(2)N-glycylglycylglycine-BBN[7-14]NH(2) peptide with the following general sequence: H(2)N-G-G-G-Q-W-A-V-G-H-L-M-(NH(2)). This conjugate was purified by reversed-phase high-performance liquid chromatography and characterized by electrospray-ionization mass spectra. The fluorescent probe Alexa Fluor 680-G-G-G-BBN[7-14]NH(2) conjugate was prepared by reaction of Alexa Fluor 680 succinimidyl ester to H(2)N-G-G-G-BBN[7-14]NH(2) in dimethylformamide (DMF). In vitro competitive binding assays, using (125)I-Tyr(4)-BBN as the radiolabeling gold standard, demonstrated an inhibitory concentration 50% value of 7.7 +/- 1.4 nM in human T-47D breast cancer cells. Confocal fluorescence microscopy images of Alexa Fluor 680-G-G-G-BBN[7-14]NH(2) in human T-47D breast cancer cells indicated specific uptake, internalization, and receptor blocking of the fluorescent bioprobe in vitro. In vivo investigations in SCID mice bearing xenografted T-47D breast cancer lesions demonstrated the ability of this new conjugate to specifically target tumor tissue with high selectivity and affinity.     

The application of anti‐ESAT‐6 monoclonal antibody fluorescent probe in ex vivo near‐infrared fluorescence imaging in mice with pulmonary tuberculosis

Here, we aimed to assess the feasibility of anti‐ESAT‐6 monoclonal antibody (mAb) coupling with IR783 and rhodamine fluorescent probe in the detection of ESAT‐6 expression in tuberculosis tissue of mice using near‐infrared fluorescence imaging. IR783 and rhodamine were conjugated to the anti‐ESAT‐6 mAb or IgG. Mice in the experimental group were injected with fluorescence‐labeled mAb probe, and mice in the control group were injected with fluorescence‐labeled non‐specific IgG antibody. Twenty‐four hours later, the lung tissue of mice was examined using ex vivo near‐infrared fluorescence imaging. In addition, the contrast‐to‐noise ratio (CNR) was calculated by measuring the signal intensities of the pulmonary lesions, normal lung tissue and background noise. The frozen lung tissue section was examined under fluorescence microscopy and compared with hemoxylin and eosin (HE) staining. The ex vivo near‐infrared fluorescence imaging showed that the fluorescence signal in the lung tuberculosis lesions in the experimental group was significantly enhanced, whereas there was only a weak fluorescence signal or even no fluorescence signal in the control group. CNR values were 64.40 ± 7.02 (n = 6) and 8.75 ± 3.87 (n = 6), respectively (t = 17.01, p < 0.001). The fluorescence accumulation distribution detected under fluorescence microscopy was consistent with HE staining of the tuberculosis region. In conclusion, anti‐ESAT‐6 mAb fluorescent probe could target and be applied in specific ex vivo imaging of mice tuberculosis, and may be of further use in tuberculosis in living mice. Copyright © 2013 John Wiley & Sons, Ltd.     

重组腺相关病毒小鼠骨骼肌中近红外荧光蛋白表达及活体成像

近红外荧光蛋白因激发光和发射光波长位于近红外区,在动物组织中光吸收和光散射最低,更适宜于动物活体组织的深层成像.构建了一种携带近红外荧光蛋白(near-infrared fluorescent protein,iRFP)713基因的重组表达质粒pAAV-iRFP713,将重组表达质粒与辅助质粒共转染AAV-293细胞,包装重组腺相关病毒(recombinant adeno-associated virus,rAAV)rAAV-iRFP713.重组腺相关病毒表达载体感染体外培养的癌细胞,48h后,荧光显微镜检测显示近红外荧光蛋白在癌细胞中高效表达,荧光明亮.重组腺相关病毒表达载体注射小鼠骨骼肌,48h后,用近红外荧光活体成像系统检测证明近红外荧光蛋白在小鼠骨骼肌中表达较强, 活体组织成像清晰.实验结果表明近红外荧光蛋白在体内体外均能很好地表达并荧光成像,为动物活体组织标记和成像的研究提供新方法.     

Synthesis and biological evaluation of water-soluble progesterone-conjugated probes for magnetic resonance imaging of hormone related cancers

Progesterone receptor (PR) is strongly associated with disease prognosis and therapeutic efficacy in hormone-related diseases such as endometriosis and breast, ovarian, and uterine cancers. Receptor status is currently determined by immunohistochemistry assays. However, noninvasive PR imaging agents could improve disease detection and help elucidate pathological molecular pathways, leading to new therapies and animal disease models. A series of water-soluble PR-targeted magnetic resonance imaging (MRI) probes were synthesized using Cu(I)-catalyzed click chemistry and evaluated in vitro and in vivo. These agents demonstrated activation of PR in vitro and preferential accumulation in PR(+) compared to PR(-) human breast cancer cells with low toxicity. In xenograft tumor models, the agents demonstrated enhanced signal intensity in PR(+) tumors compared to PR(-) tumors. The results suggest that these agents may be promising MRI probes for PR(+) diseases.     

Preclinical molecular imaging of the translocator protein (TSPO) in a metastases model based on breast cancer xenografts propagated in the murine brain

Previous studies have demonstrated the feasibility of translocator protein (TSPO) imaging to visualize and quantify human breast adenocarcinoma (MDA-MB-231) cells in vivo using a TSPO-targeted near-infrared (NIR) probe (NIR-conPK11195). This study aimed to extend the use of the TSPO-targeted probe to a more biologically relevant and clinically important tumor microenvironment as well as to assess our ability to longitudinally detect the presence and progression of breast cancer cells in the brain. The in vivo biodistribution and accumulation of NIR-conPK11195 and free (unconjugated) NIR dye were quantitatively evaluated in intracranial MDA-MB-231-bearing mice and non-tumor-bearing control mice longitudinally once a week from two to five weeks post-inoculation. The in vivo time-activity curves illustrate distinct clearance profiles for NIR-conPK11195 and free NIR dye, resulting in preferential accumulation of the TSPO-targeted probe in the intracranial tumor bearing hemisphere (TBH) with significant tumor contrast over normal muscle tissue (p < 0.005 at five weeks; p < 0.01 at four weeks). In addition, the TSPO-labeled TBHs demonstrated significant contrast over the TBHs of mice injected with free NIR dye (p < 0.001 at four and five weeks) as well as over the TSPO-labeled non-tumor-bearing hemispheres (NTBHs) of control mice (p < 0.005 at four and five weeks). Overall, TSPO-targeted molecular imaging appears useful for visualizing and quantifying breast cancer xenografts propagated in the murine brain and may assist in preclinical detection, diagnosis and monitoring of metastatic disease as well as drug discovery. Furthermore, these results indicate it should be possible to perform TSPO-imaging of breast cancer cells in the brain using radiolabeled TSPO-targeted agents, particularly in light of the fact that [11C]-labeled TSPO probes such as [11C]-PK 11195 have been successfully used to image gliomas in the clinic.     

Activatable Near-Infrared Fluorescent Probe for In Vivo Imaging of Fibroblast Activation Protein-alpha

Fibroblast activation protein-alpha (FAPα) is a cell surface glycoprotein which is selectively expressed by tumor-associated fibroblasts in malignant tumors but rarely on normal tissues. FAPα has also been reported to promote tumor growth and invasion and therefore has been of increasing interest as a promising target for designing tumor-targeted drugs and imaging agents. Although medicinal study on FAPα inhibitors has led to the discovery of many FAPα-targeting inhibitors including a drug candidate in a phase II clinical trial, the development of imaging probes to monitor the expression and activity of FAPα in vivo has largely lagged behind. Herein, we report an activatable near-infrared (NIR) fluorescent probe (ANP(FAP)) for in vivo optical imaging of FAPα. The ANP(FAP) consists of a NIR dye (Cy5.5) and a quencher dye (QSY21) which are linked together by a short peptide sequence (KGPGPNQC) specific for FAPα cleavage. Because of the efficient fluorescence resonance energy transfer (FRET) between Cy5.5 and QSY21 in ANP(FAP), high contrast on the NIR fluorescence signal can be achieved after the cleavage of the peptide sequence by FAPα both in vitro and in vivo. In vitro assay on ANP(FAP) indicated the specificity of the probe to FAPα. The in vivo optical imaging using ANP(FAP) showed fast tumor uptake as well as high tumor to background contrast on U87MG tumor models with FAPα expression, while much lower signal and tumor contrast were observed in the C6 tumor without FAPα expression, demonstrating the in vivo targeting specificity of the ANP(FAP). Ex vivo imaging also demonstrated ANP(FAP) had high tumor uptake at 4 h post injection. Collectively, these results indicated that ANP(FAP) could serve as a useful NIR optical probe for early detection of FAPα expressing tumors.     

Novel α(2)β(1) integrin-targeted peptide probes for prostate cancer imaging

Accumulating experimental evidence indicates that overexpression of α(2)β(1) integrin may correlate with progression in human prostate cancer. The objective of this study was to design a novel imaging probe based on the Asp-Gly-Glu-Ala (DGEA) peptide for near-infrared-fluorescent (NIRF) imaging of α(2)β(1) integrin expression in prostate cancer. The peptides were conjugated with appropriate fluorescent dyes, and the binding affinity of these probes was evaluated by flow cytometry in three human prostate cell lines (PC-3, CWR-22, and LNCaP). In vivo NIRF imaging of the α(2)β(1)-positive PC-3 xenograft model was performed to evaluate the α(2)β(1) targeted probe. In vitro immunofluorescence staining was carried out to confirm the α(2)β(1) integrin expression level. Flow cytometry analysis showed that PC-3 had the highest probe uptake, followed by CWR-22 and LNCaP tumor cells. In the subcutaneous PC-3 model, the tumor demonstrated prominent uptake with good tumor to background contrast. Immunohistochemistry staining also supported the in vivo optical imaging results. DGEA-based optical agents have been developed for specific imaging of α(2)β(1) integrin expression. In vitro and in vivo localization demonstrated the potential of this agent to identify tumor subtypes amenable to anti-α(2)β(1) integrin treatment and potentially provide prognostic information regarding tumor progression.     

Novel cancer-targeting SPECT/NIRF dual-modality imaging probe 99mTc-PC-1007: Synthesis and biological evaluation

Synthesis, characterization, in vitro and in vivo biological evaluation of a heptamethine cyanine based dual-mode single-photon emission computed tomography (SPECT)/near infrared fluorescence (NIRF) imaging probe ^99mTc-PC-1007 is described. ^99mTc-PC-1007 exhibited preferential accumulation in human breast cancer MCF-7 cells. Cancer-specific SPECT/CT and NIRF imaging of ^99mTc-PC-1007 was performed in a breast cancer xenograft model. The probe uptake ratio of tumor to control (spinal cord) was calculated to be 4.02 ± 0.56 at 6 h post injection (pi) and 8.50 ± 1.41 at 20 h pi (P < 0.0001). Pharmacokinetic parameters such as blood clearance and organ distribution were assessed.     

Fast multispectral optoacoustic tomography (MSOT) for dynamic imaging of pharmacokinetics and biodistribution in multiple organs

The characterization of pharmacokinetic and biodistribution profiles is an essential step in the development process of new candidate drugs or imaging agents. Simultaneously, the assessment of organ function related to the uptake and clearance of drugs is of great importance. To this end, we demonstrate an imaging platform capable of high-rate characterization of the dynamics of fluorescent agents in multiple organs using multispectral optoacoustic tomography (MSOT). A spatial resolution of approximately 150 μm through mouse cross-sections allowed us to image blood vessels, the kidneys, the liver and the gall bladder. In particular, MSOT was employed to characterize the removal of indocyanine green from the systemic circulation and its time-resolved uptake in the liver and gallbladder. Furthermore, it was possible to track the uptake of a carboxylate dye in separate regions of the kidneys. The results demonstrate the acquisition of agent concentration metrics at rates of 10 samples per second at a single wavelength and 17 s per multispectral sample with 10 signal averages at each of 5 wavelengths. Overall, such imaging performance introduces previously undocumented capabilities of fast, high resolution in vivo imaging of the fate of optical agents for drug discovery and basic biological research.     

Src/FAK-mediated regulation of E-cadherin as a mechanism for controlling collective cell movement: Insights from in vivo imaging

Recent advances in confocal and multi-photon microscopy, together with fluorescent probe development, have enabled cancer biology studies to go beyond the culture dish and interrogate cancer-associated processes in the complex in vivo environment. Regulation of the tumor suppressor protein E-cadherin plays an important role in cancer development and progression, and may contribute to the decision between ‘single cell’ and ‘collective invasion’ in vivo. Mounting evidence from in vitro and in vivo experiments places the two nonreceptor protein tyrosine kinases Src and Focal Adhesion Kinase at the heart of E-cadherin regulation and the crosstalk between integrins and cadherins. Here we discuss recent insights, attained using high-resolution fluorescent in vivo imaging, into the regulation of E-cadherin and collective invasion. We focus on the regulatory crosstalk between the Src/FAK signaling axis and E-cadherin in vivo.Key words: Src, FAK, E-cadherin, EMT, fluorescent in vivo imaging     

Nanoconjugate based on polymalic acid for tumor targeting

A new prototype of polymer-derived drug delivery system, the nanoconjugate Polycefin, was tested for its ability to accumulate in tumors based on enhanced permeability and retention (EPR) effect and receptor mediated endocytosis. Polycefin was synthesized for targeted delivery of Morpholino antisense oligonucleotides into certain tumors. It consists of units that are covalently conjugated with poly(beta-l-malic acid) (M(w) 50,000, M(w)/M(n) 1.3) highly purified from cultures of myxomycete Physarum polycephalum. The units are active in endosomal uptake, disruption of endosomal membranes, oligonucleotide release in the cytoplasm, and protection against enzymatic degradation in the vascular system. The polymer is biodegradable, non-immunogenic and non-toxic. Polycefin was also coupled with AlexaFluor 680 C2-maleimide dye for in vivo detection. Nude mice received subcutaneous injections of MDA-MB 468 human breast cancer cells into the left posterior mid-dorsum or intracranial injections of human glioma cell line U87MG. Polycefin at concentration of 2.5mg/kg was injected via the tail vein. In vivo fluorescence tumor imaging was performed at different time points, 0-180 min up to 24h after the drug injection. The custom-made macro-illumination imaging MISTI system was used to examine the in vivo drug accumulation in animals bearing human breast and brain tumors. In breast tumors the fluorescence signal in large blood vessels and in the tumor increased rapidly until 60 min and remained in the tumor at a level 6 times higher than in non-tumor tissue (180 min) (p<0.003). In brain tumors drug accumulated selectively in 24h without any detectable signal in non-tumor areas. The results of live imaging were corroborated histologically by fluorescence microscopic examination of various organs. In addition to tumors, only kidney and liver showed some fluorescent signal.     

A Versatile Technique for the In Vivo Imaging of Human Tumor Xenografts Using Near-Infrared Fluorochrome-Conjugated Macromolecule Probes

Here, we present a versatile method for detecting human tumor xenografts in vivo, based on the enhanced permeability and retention (EPR) effect, using near-infrared (NIR) fluorochrome-conjugated macromolecule probes. Bovine serum albumin (BSA) and two immunoglobulins—an anti-human leukocyte antigen (HLA) monoclonal antibody and isotype control IgG_2a—were labeled with XenoLight CF770 fluorochrome and used as NIR-conjugated macromolecule probes to study whole-body imaging in a variety of xenotransplantation mouse models. NIR fluorescent signals were observed in subcutaneously transplanted BxPC-3 (human pancreatic cancer) cells and HCT 116 (colorectal cancer) cells within 24 h of NIR-macromolecule probe injection, but the signal from the fluorochrome itself or from the NIR-conjugated small molecule (glycine) injection was not observed. The accuracy of tumor targeting was confirmed by the localization of the NIR-conjugated immunoglobulin within the T-HCT 116 xenograft (in which the orange-red fluorescent protein tdTomato was stably expressed by HCT 116 cells) in the subcutaneous transplantation model. However, there was no significant difference in the NIR signal intensity of the region of interest between the anti-HLA antibody group and the isotype control group in the subcutaneous transplantation model. Therefore, the antibody accumulation within the tumor in vivo is based on the EPR effect. The liver metastasis generated by an intrasplenic injection of T-HCT 116 cells was clearly visualized by the NIR-conjugated anti-HLA probe but not by the orange-red fluorescent signal derived from the tdTomato reporter. This result demonstrated the superiority of the NIR probes over the tdTomato reporter protein at enhancing tissue penetration. In another xenograft model, patient-derived xenografts (PDX) of LC11-JCK (human non-small cell lung cancer) were successfully visualized using the NIR-conjugated macromolecule probe without any genetic modification. These results suggested that NIR-conjugated macromolecule, preferably, anti-HLA antibody probe is a valuable tool for the detection of human tumors in experimental metastasis models using whole-body imaging.     

A comparison of the emission efficiency of four common green fluorescence dyes after internalization into cancer cells

In vivo optical imaging to enhance the detection of cancer during endoscopy or surgery requires a targeted fluorescent probe with high emission efficiency and high signal-to-background ratio. One strategy to accurately detect cancers is to have the fluorophore internalize within the cancer cells permitting nonbound fluorophores to be washed away or absorbed. The choice of fluorophores for this task must be carefully considered. For depth of penetration, near-infrared probes are ordinarily preferred but suffer from relatively low quantum efficiency. Although green fluorescent protein has been widely used to image tumors on internal organs in mice, green fluorescent probes are better suited for imaging the superficial tissues because of the short penetration distance of green light in tissue and the highly efficient production of signal. While the fluorescence properties of green fluorophores are well-known in vitro, less attention has been paid to their fluorescence once they are internalized within cells. In this study, the emission efficiency after cellular internalization of four common green fluorophores conjugated to avidin (Av-fluorescein, Av-Oregon green, Av-BODIPY-FL, and Av-rhodamine green) were compared after each conjugate was incubated with SHIN3 ovarian cancer cells. Using the lectin binding receptor system, the avidin-fluorophore conjugates were endocytosed, and their fluorescence was evaluated with fluorescence microscopy and flow cytometry. While fluorescein demonstrated the highest signal outside the cell, among the four fluorophores, internalized Av-rhodamine green emitted the most light from SHIN3 ovarian cancer cells both in vitro and in vivo. The internalized Av-rhodamine green complex appeared to localize to the endoplasmic vesicles. Thus, among the four common green fluorescent dyes, rhodamine green is the brightest green fluorescence probe after cellular internalization. This information could have implications for the design of tumor-targeted fluorescent probes that rely on cellular internalization for cancer detection.     

Determination of optimal rhodamine fluorophore for in vivo optical imaging

Optical imaging has the potential to improve the efficacy of surgical and endoscopic approaches to cancer treatment; however, the optimal type of fluorescent probe has not yet been established. It is well-known that rhodamine-core-derived fluorophores offer a combination of desirable properties such as good photostability, high extinction coefficient, and high fluorescence quantum yield. However, despite the ubiquitous use of rhodamine fluorophores for in vivo optical imaging, it remains to be determined if unique chemical properties among individual rhodamine core family members affect fluorophore parameters critical to in vivo optical imaging applications. These parameters include preserved fluorescence intensity in low pH environments, similar to that of the endolysosome; efficient fluorescence signal despite conformational changes to targeting proteins as may occur in harsh subcellular environments; persistence of fluorescence after cellular internalization; and sufficient signal-to-background ratios to permit the identification of fluorophore-targeted tumors. In the present study, we conjugated 4 common rhodamine-core based fluorescent dyes to a clinically feasible and quickly internalizing D-galactose receptor targeting reagent, galactosamine serum albumin (GmSA), and conducted a series of in vitro and in vivo experiments using a metastatic ovarian cancer mouse model to determine if differences in optical imaging properties exist among rhodamine fluorophores and if so, which rhodamine core possesses optimal characteristics for in vivo imaging applications. Herein, we demonstrate that the rhodamine-fluorophore, TAMRA, is the most robust of the 4 common rhodamine fluorophores for in vivo optical imaging of ovarian cancer metastases to the peritoneum.     

Near-infrared pH-activatable fluorescent probes for imaging primary and metastatic breast tumors

Highly tumor selective near-infrared (NIR) pH-activatable probe was developed by conjugating pH-sensitive cyanine dye to a cyclic arginine-glycine-aspartic acid (cRGD) peptide targeting α(v)β(3) integrin (ABIR), a protein that is highly overexpressed in endothelial cells during tumor angiogenesis. The NIR pH-sensitive dye used to construct the probe exhibits high spectral sensitivity with pH changes. It has negligible fluorescence above pH 6 but becomes highly fluorescent below pH 5, with a pK(a) of 4.7. This probe is ideal for imaging acidic cell organelles such as tumor lysosomes or late endosomes. Cell microscopy data demonstrate that binding of the cRGD probe to ABIR facilitated the endocytosis-mediated lysosomal accumulation and subsequent fluorescence enhancement of the NIR pH-activatable dye in tumor cells (MDA-MB-435 and 4T1/luc). A similar fluorescence enhancement mechanism was observed in vivo, where the tumors were evident within 4 h post injection. Moreover, lung metastases were also visualized in an orthotopic tumor mouse model using this probe, which was further confirmed by histologic analysis. These results demonstrate the potential of using the new integrin-targeted pH-sensitive probe for the detection of primary and metastatic cancer.     

Synthesis and evaluation of near-infrared fluorescent sulfonamide derivatives for imaging of hypoxia-induced carbonic anhydrase IX expression in tumors

A series of human carbonic anhydrase (hCA) IX inhibitors conjugated to various near-infrared fluorescent dyes was synthesized with the aim of imaging hypoxia-induced hCA IX expression in tumor cells in vitro, ex vivo and in vivo. The resulting compounds were profiled for inhibition of transmembrane hCA IX showing a range of potencies from 7.5 to 116 nM and up to 50-fold selectivity over the cytosolic form hCA II. Some of the compounds also showed inhibition selectivity for other transmembrane forms hCA XII and XIV as well. Compounds incubated in vitro with HeLa cells cultured under normoxic and hypoxic conditions detected upregulation of hCA IX under hypoxia by fluorescence microscopy. A pilot in vivo study in HT-29 tumor bearing mice showed significant accumulation of a fluorescent acetazolamide derivative in tumor tissue with little accumulation in other tissues. Approximately 10% of injected dose was non-invasively quantified in tumors by fluorescence molecular tomography (FMT), demonstrating the promise of these new compounds for quantitative imaging of hCA IX upregulation in live animals.     

In Vivo Imaging and Quantification of Carbonic Anhydrase IX Expression as an Endogenous Biomarker of Tumor Hypoxia

Carbonic anhydrase IX (CA IX) is a transmembrane protein that has been shown to be greatly upregulated under conditions of hypoxia in many tumor cell lines. Tumor hypoxia is associated with impaired efficacy of cancer therapies making CA IX a valuable target for preclinical and diagnostic imaging. We have developed a quantitative in vivo optical imaging method for detection of CA IX as a marker of tumor hypoxia based on a near-infrared (NIR) fluorescent derivative of the CA IX inhibitor acetazolamide (AZ). The agent (HS680) showed single digit nanomolar inhibition of CA IX as well as selectivity over other CA isoforms and demonstrated up to 25-fold upregulation of fluorescent CA IX signal in hypoxic versus normoxic cells, which could be blocked by 60%–70% with unlabeled AZ. CA IX negative cell lines (HCT-116 and MDA-MB-231), as well as a non-binding control agent on CA IX positive cells, showed low fluorescent signal under both conditions. In vivo FMT imaging showed tumor accumulation and excellent tumor definition from 6–24 hours. In vivo selectivity was confirmed by pretreatment of the mice with unlabeled AZ resulting in >65% signal inhibition. HS680 tumor signal was further upregulated >2X in tumors by maintaining tumor-bearing mice in a low oxygen (8%) atmosphere. Importantly, intravenously injected HS680 signal was co-localized specifically with both CA IX antibody and pimonidazole (Pimo), and was located away from non-hypoxic regions indicated by a Hoechst stain. Thus, we have established a spatial correlation of fluorescence signal obtained by non-invasive, tomographic imaging of HS680 with regions of hypoxia and CA IX expression. These results illustrate the potential of HS680 and combined with FMT imaging to non-invasively quantify CA IX expression as a hypoxia biomarker, crucial to the study of the underlying biology of hypoxic tumors and the development and monitoring of novel anti-cancer therapies.     

Development of high-sensitivity near-infrared fluorescence imaging device for early cancer detection

We have developed a high-sensitivity near-infrared (NIR) optical imaging system for noninvasive cancer detection based on the molecular-labeled fluorescent contrast agents. Recent developments in molecular beacons offer a way to selectively tag various precancer and cancer signatures and provide high tumor-to-background contrast. Near-infrared imaging can deeply probe tissue up to a couple of centimeters; thus, it possesses the potential for noninvasive detection of breast or lymph node cancer. A phase cancellation (in- and antiphase) device is used to increase the sensitivity in detecting fluorescent photons and the accuracy of tumor localization. The optoelectronic system consists of the laser diode sources, fiber optics, interference filter (to select the fluorescent photons), and the high-sensitivity photon detector (photomultiplier tube). The source-detector pair scans the tissue surface in multiple directions, and the localization image can be obtained by angular back-projection reconstruction. Simulations and experimental data demonstrated the feasibility of detection and localization offluorescent object embedded inside the highly scattering media. Tumor-bearing mouse model with injection of fluorescent contrast agents is used to simulate the human breast tumor labeled with molecular beacons. The system can detect fluorescent contrast agents as small as one nanomole at the depth of three centimeters, with a three-millimeter localization error. This instrument has the potential for tumor diagnosis and imaging, and the accuracy of the localization suggests that this system could help guide the clinical fine-needle biopsy. Also, this portable device would be complementary to x-ray mammography and provide add-on information on early diagnosis and localization of breast tumor.