90Y and 111In complexes of a DOTA-conjugated integrin alpha v beta 3 receptor antagonist: different but biologically equivalent

90Y-TA138 is a (90)Y-labeled nonpeptide integrin alpha(v)beta(3) receptor antagonist that binds with high affinity and specificity to integrin alpha(v)beta(3) receptors overexpressed on both endothelial and tumor cells. (90)Y-TA138 has demonstrated significant therapeutic effects in several preclinical tumor-bearing animal models. Since (90)Y is a pure beta-emitter, (111)In-TA138 has been chosen as the imaging surrogate for dosimetry determination of (90)Y-TA138. This report describes the synthesis of (111)In-TA138 and biological evaluations of both (111)In-TA138 and (90)Y-TA138 in the c-neu Oncomouse model. The HPLC data shows that (111)In-TA138 is more hydrophilic with the retention time approximately 4.5 min shorter than that of (90)Y-TA138 under identical chromatographic conditions. Since the only difference between (111)In-TA138 and (90)Y-TA138 is the metal ion, the HPLC retention time difference strongly suggests that indium and yttrium chelates do not share the same coordination sphere in solution even though they are coordinated by the same DOTA conjugate. Despite their differences in lipophilicity and solution structure, biodistribution data in the c-neu Oncomouse model clearly showed that (111)In-TA138 and (90)Y-TA138 are biologically equivalent with respect to their uptake in tumors and other major organs. Therefore, (111)In-TA138 is useful as an imaging surrogate for (90)Y-TA138 and should be able to predict the radiation dosimetry of (90)Y-TA138, a therapeutic radiopharmaceutical for treatment of rapidly growing tumors.     

Structure-activity relationships of 111In- and 99mTc-labeled quinolin-4-one peptidomimetics as ligands for the vitronectin receptor: potential tumor imaging agents

The integrin receptor alpha(v)beta(3) is overexpressed on the endothelial cells of growing tumors and on some tumor cells themselves. Radiolabeled alpha(v)beta(3) antagonists have demonstrated potential application as tumor imaging agents and as radiotherapeutic agents. This report describes the total synthesis of eight new HYNIC and DOTA conjugates of receptor alpha(v)beta(3) antagonists belonging to the quinolin-4-one class of peptidomimetics, and their radiolabeling with (99m)Tc (for HYNIC) and (111)In (for DOTA). Tethering of the radionuclide-chelator complexes was achieved at two different sites on the quinolin-4-one molecule. All such derivatives maintained high affinity for receptor alpha(v)beta(3) and high selectivity versus receptors alpha(IIb)beta(3), alpha(v)beta(5), alpha(5)beta(1). Biodistribution of the radiolabeled compounds was evaluated in the c-neu Oncomouse mammary adenocarcinoma model. DOTA conjugate (111)In-TA138 presented the best biodistribution profile. Tumor uptake at 2 h postinjection was 9.39% of injected dose/g of tissue (%ID/g). Activity levels in selected organs was as follows: blood, 0.54% ID/g; liver, 1.94% ID/g; kidney, 2.33% ID/g; lung, 2.74% ID/g; bone, 1.56% ID/g. A complete biodistribution analysis of (111)In-TA138 and the other radiolabeled compounds of this study are presented and discussed. A scintigraphic imaging study with (111)In-TA138 showed a clear delineation of the tumors and rapid clearance of activity from nontarget tissues.     

In vitro and in vivo evaluation of a novel 99mTc(CO)3-pyrazolyl conjugate of cyclo-(Arg-Gly-Asp-d-Tyr-Lys)

Radiolabeled peptides containing the Arg-Gly-Asp amino acid sequence (single letter code = RGD) have been studied extensively to target integrin receptors upregulated on tumor cells and neovasculature. Integrins are cell surface transmembrane glycoproteins that exist as alphabeta heterodimers. The alphavbeta3 integrin is known to be overexpressed in many tumor types and is expressed at lower levels in normal tissues. Furthermore, alphavbeta3 and alphavbeta5 subtypes are expressed in neovasculature during angiogenesis. Thus, there is some impetus to image angiogenesis and tumor formation in vivo using RGD-based peptide targeting vectors. In this study, we report the design and development of a new cyclic RGD analogue cyclo-[Arg-Gly-Asp-d-Tyr-Lys(PZ)] (PZ = 3,5-Me2-pz(CH2)2N((CH2)3COOH)(CH2)2NH2) that can be radiolabeled with the [99mTc(CO)3(H2O)3]+ metal aquaion. Radiochemical evaluation of this new conjugate in vitro indicated a facile radiosynthesis of the new 99mTc-RGD conjugate with high radiolabeling yields (>or=95%) and high specific activities. In vitro internalization and blocking assays in alphavbeta3 receptor-positive, human M21 melanoma cancer cells showed the ability of this conjugate to target the integrin receptor with high specificity and selectivity. In vivo pharmacokinetic studies in normal CF-1 mice showed rapid clearance from blood with excretion primarily via/through the renal-urinary system. In vivo accumulation of radioactivity in mice bearing either alphavbeta3 receptor-positive or negative human melanoma tumors showed receptor specific uptake of tracer with accumulations of 2.50 +/- 0.29 and 0.71 +/- 0.08% ID/g in alphavbeta3 integrin positive (M21) and negative (M21L) tumors at 1 h postinjection (p.i.), respectively.     

Use of antibody as carrier of oligomers of peptidomimetic alphavbeta3 antagonist to target tumor-induced neovasculature

Sulfhydryl selective reactions were explored to conjugate oligomers of a peptidomimetic integrin alphavbeta3 antagonist, 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-(S)-aminoethylsulfonylamino-beta-alanine (IA) to monoclonal antibody (MoAb) to increase integrin alphavbeta3 receptor-binding avidity. To generate sulfhydryl groups, N-succinimidyl-S-acetylthioacetate (SATA) was conjugated to both MoAb and IA. Sulfhydryl groups were then generated upon the deacetylation of the protecting acetyl group from the S-acetylthioacetato (ATA) moiety of MoAb-(ATA)n or IA-ATA with 0.02 M hydroxylamine in the presence of 1 mM EDTA at pH 7.2. The major focus was on optimizing the reaction concentrations, molar ratios, and reaction pH to conjugate high levels of IA-(A-SH) to MoAb-(A-SH)n without causing the inter- and intramolecular cross-linking of MoAb. Stepwise reactions of MoAb-(A-SH)n (15 microM MoAb) with a homobifunctional cross-linker, 1,8-bis(maleimido)diethylene glycol (BM[PEO]2) at a >50x molar excess to the -SH, followed by the reaction of the purified product MoAb-(A-S-succinimidomaleimido-[PEO]2)n with IA-(A-SH) at pH 7.2 afforded monomeric MoAb-(A-S-succinimido-[PEO]2-succinimido-S-A-IA)n with <10% high molecular weight oligomeric MoAb. Monomeric MoAb-(A-S-S-[PEO]2-S-S-A-IA)10 (MoAb-IA10) radiolabeled with 111In using 2-(p-isothiocyanatobenzyl)cyclohexyl-DTPA and with 125I using the Iodogen method showed >70% bindability to 0.4 microM alphavbeta3. When injected iv to nude mice with the receptor-positive M21 tumor, MoAb-IA10 radiolabeled with both 111In and 125I accumulated rapidly and was retained in the tumor for a 44 h period while the radioactivity cleared rapidly from the blood, thereby resulting in increasing tumor-to-blood ratios over time. The tumor uptake was similar between the 125I label and the 111In label for a 44 h period. In contrast, the blood radioactivity was lower, but liver and other organ uptakes were much higher for the 111In label than for the 125I. The 111In label produced higher tumor-to-blood ratios but much lower tumor-to-organ ratios than the 125I. The rapid blood clearance, a short peak tumor uptake time, and a low peak tumor uptake value with prolonged tumor retention of this macromolecule appear to support a hypothesis that MoAb-IA10 primarily binds to alphavbeta3 receptors on angiogenic vessels, but not on the tumor. This hypothesis was substantiated by the fluorescence microscopic analysis of FITC-MoAb-IA10, which showed that FITC-MoAb-IA10 outlined neovasculatures but not tumor cells at 4 and 21 h ex vivo. Additional proof was observed when blood vessels outlined with rhodamine-lectin, which specifically binds to blood vessels, were superimposable on neovasculatures outlined with FITC-MoAb-IA10.     

Evaluation of a (99m)Tc-labeled cyclic RGD tetramer for noninvasive imaging integrin alpha(v)beta3-positive breast cancer

Integrin alphavbeta3 plays a critical role in tumor angiogenesis and metastasis. Radiolabeled RGD peptides that are integrin alphavbeta3-specific are very useful for noninvasive imaging of integrin expression in rapidly growing and metastatic tumors. In this study, we determined the binding affinity of E{E[c(RGDfK)]2}2 (tetramer) and its 6-hydrazinonicotinamide conjugate (HYNIC-tetramer) against the binding of 125I-echistatin to the integrin alphavbeta3-positive MDA-MB-435 breast cancer cells. The athymic nude mice bearing MDA-MB-435 xenografts were used to evaluate the potential of ternary ligand complex [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] (TPPTS = trisodium triphenylphosphine-3,3',3' '-trisulfonate) as a new radiotracer for imaging breast cancer integrin alphavbeta3 expression by single photon emission computed tomography (SPECT). It was found that the binding affinity of tetramer (IC50 = 51 +/- 11 nM) was slightly higher than that of its dimeric analogue (IC50 = 78 +/- 27 nM) and is comparable to that of the HYNIC-tetramer conjugate (IC50 = 55 +/- 11 nM) within the experimental error. Biodistribution data showed that [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] had a rapid blood clearance (4.61 +/- 0.81 %ID/g at 5 min postinjection (p.i.) and 0.56 +/- 0.12 %ID/g at 120 min p.i.) and was excreted mainly via the renal route. [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] had high tumor uptake with a long tumor retention (5.60 +/- 0.87 %ID/g and 7.30 +/- 1.32 %ID/g at 5 and 120 min p.i., respectively). The integrin alphavbeta3-specificity was demonstrated by co-injection of excess E[c(RGDfK)]2, which resulted in a significant reduction in tumor uptake of the radiotracer. The metabolic stability of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] was determined by analyzing urine and feces samples from the tumor-bearing mice at 120 min p.i. In the urine, about 20% of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] remained intact while only approximately 15% metabolized species was detected in feces. SPECT images displayed significant radiotracer localization in tumor with good contrast as early as 1 h p.i. The high tumor uptake and fast renal excretion make [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] a promising radiotracer for noninvasive imaging of the integrin alphavbeta3-positive tumors by SPECT.     

Switch from alphavbeta5 to alphavbeta6 integrin expression protects squamous cell carcinomas from anoikis

Stratified squamous epithelia express the alphavbeta5 integrin, but in squamous cell carcinomas (SCCs) there is down-regulation of alphavbeta5 and up-regulation of alphavbeta6. To investigate the significance of this finding, we transduced an alphav-negative human SCC line with retroviral vectors encoding alphav integrins. alphavbeta5-expressing cells underwent suspension-induced apoptosis (anoikis), whereas alphav-negative cells and cells expressing alphavbeta6 did not. Resistance to anoikis correlated with PKB/Akt activation in suspension, but not with changes in PTEN or p110alpha PI3 kinase levels. Anoikis was induced in parental and alphavbeta6-expressing cells by inhibiting PI3 kinase. Conversely, activation of Akt or inhibition of caspases in alphavbeta5-expressing cells suppressed anoikis. Caspase inhibition resulted in increased phosphoAkt, placing caspase activation upstream of decreased Akt activation. Anoikis required the cytoplasmic domain of beta5 and was independent of the death receptor pathway. These results suggest that down-regulation of alphavbeta5 through up-regulation of alphavbeta6 may protect SCCs from anoikis by activating an Akt survival signal.     

Role of phosphatidylinositol 3-kinase in the binding of Bordetella pertussis to human monocytes

Bordetella pertussis, the causative agent of whooping cough, adheres to human monocytes by means of filamentous haemagglutinin (FHA), a bacterial surface protein that is recognized by complement receptor type 3 (CR3, alphaMbeta2 integrin). Previous work has shown that an FHA Arg-Gly-Asp (RGD, residues 1097-1099) site interacts with a complex composed of leucocyte response integrin (LRI, alphavbeta3 integrin) and integrin-associated protein (IAP, CD47) on human monocytes, resulting in enhancement of CR3-mediated bacterial binding. However, the pathway that mediates alphavbeta3-alphaMbeta2 integrin signalling remains to be characterized. Here we describe the involvement of phosphatidylinositol 3-kinase (PI3-K) in this pathway. Wortmannin and LY294002, inhibitors of PI3-K, reduced alphavbeta3/IAP-upregulated, CR3-associated bacterial binding to human monocytes. B. pertussis infection of human monocytes resulted in a marked recruitment of cellular PI3-K to the sites of B. pertussis contact. In contrast, cells infected with an isogenic strain carrying a G1098A mutation at the FHA RGD site did not show any recruitment of PI3-K. We found that ligation of FHA by alphavbeta3/IAP induced RGD-dependent tyrosine phosphorylation of a 60 kDa protein, which associated with IAP and PI3-K in human monocytes. These results suggest that PI3-K and a tyrosine phosphorylated 60 kDa protein may be involved in this biologically important integrin signalling pathway.     

Design, synthesis, and biological evaluation of an antagonist-bombesin analogue as targeting vector

The gastrin releasing peptide receptor (GRP-R) is overexpressed on a number of tumors and cancer cell lines including pancreas, prostate, breast, gastrointestinal, and small cell lung cancer (SCLC). Radiolabeled bombesin (BBN) analogues have exhibited high binding affinity and specificity to the GRP-R. A bombesin analogue with an antagonist targeting vector at the C-terminus, DOTA-aminohexanoyl-[D-Phe(6), Leu-NHCH 2CH 2CH3(13), des Met(14)] BBN[6-14] (1, "Bomproamide"), has been synthesized and displays high binding affinity (IC50 = 1.36 +/- 0.09 nM) against (125)I-Tyr (4)-BBN in in vitro competitive assays using PC-3 cells. Maximum internalization of (111)In-1 reached 14% in PC-3 cells after 45 min of incubation. Rapid (0.25 h PI) and high (12.21 +/- 3.2%ID/g) pancreatic uptake of (111)In-1 was observed in healthy CF-1 mice, and 90% of the activity was blocked by coinjection of 100 mug of BBN. Rapid (0.25 h PI) and high uptake (6.90 +/- 1.06%ID/g) was observed in PC-3 prostate cancer xenografts in SCID mice, as well as visualized clearly in a SPECT/CT study. These results support the use of a bombesin construct with an antagonist C-terminal vector as a candidate of choice for specific in vivo imaging of tumors overexpressing GRP-receptors.     

Integrin alpha3beta1, a novel receptor for alpha3(IV) noncollagenous domain and a trans-dominant Inhibitor for integrin alphavbeta3

Exogenous soluble human alpha3 noncollagenous (NC1) domain of collagen IV inhibits angiogenesis and tumor growth. These biological functions are attributed to the binding of alpha3NC1 to integrin alphavbeta3. However, in some tumor cells that express integrin alphavbeta3, the alpha3NC1 domain does not inhibit proliferation, suggesting that integrin alphavbeta3 expression is not sufficient to mediate the anti-tumorigenic activity of this domain. Therefore, in the present study, we searched for novel binding receptors for the soluble alpha3NC1 domain in cells lacking alphavbeta3 integrin. In these cells, soluble alpha3NC1 bound integrin alpha3beta1; however, unlike alphavbeta3, alpha3beta1 integrin did not mediate cell adhesion to immobilized alpha3NC1 domain. Interestingly, in cells lacking integrin alpha3beta1, adhesion to the alpha3NC1 domain was enhanced due to activation of integrin alphavbeta3. These findings indicate that integrin alpha3beta1 is a receptor for the alpha3NC1 domain and transdominantly inhibits integrin alphavbeta3 activation. Thus integrin alpha3beta1, in conjunction with integrin alphavbeta3, modulates cellular responses to the alpha3NC1 domain, which may be pivotal in the mechanism underpinning its anti-angiogenic and anti-tumorigenic activities.     

Near-infrared fluorescent divalent RGD ligand for integrin α(v)β(3)-targeted optical imaging

A new near-infrared fluorescent compound containing two cyclic RGD motifs, cypate-[c(RGDfK)](2) (1), was synthesized based on a carbocyanine fluorophore bearing two carboxylic acid groups (cypate) for integrin α(v)β(3)-targeting. Compared with its monovalent counterpart cypate-c(RGDfK) (2), 1 exhibited remarkable improvements in integrin α(v)β(3) binding affinity and tumor uptake in nude mice of A549. The results suggest that cypate-linked divalent ligands can serve as an important molecular platform for exploring receptor-targeted optical imaging and treatment of various diseases.     

Engineered fibronectin type III domain with a RGDWXE sequence binds with enhanced affinity and specificity to human alphavbeta3 integrin

Fibronectin is an extracellular matrix protein with broad binding specificity to cell surface receptors, integrins. The tenth fibronectin type III domain (FNfn10) is a small, autonomous domain of fibronectin containing the RGE sequence that is directly involved in integrin binding. However, in isolation FNfn10 only weakly bind to integrins. We reasoned that high-affinity and high-specificity variants of FNfn10 to a particular integrin could be engineered by optimizing residues surrounding the integrin-binding RGD sequence in the flexible FG loop. Affinity maturation of FNfn10 to alphavbeta3 integrin, an integrin up-regulated in angiogenic endothelial cells and in some metastatic tumor cells, yielded alphavbeta3-binding FNfn10 mutants with a novel RGDWXE consensus sequence. We characterized one of the RGDWXE-modified clones, FNfn10-3JCLI4, as purified protein. FNfn10-3JCLI4 binds with high affinity and specificity to purified alphavbeta3 integrin. Alanine scanning mutagenesis suggested that both the tryptophan and glutamic acid residues following the RGD sequence are required for maximal affinity and specificity for alphavbeta3. FNfn10-3JCLI4 specifically stained alphavbeta3-positive cells as detected with flow cytometry and it inhibited alphavbeta3-dependent cell adhesion. As with the anti-alphavbeta3 antibody LM609, FNfn10-3JCLI4 can interfere with in vitro capillary formation. Taken together, these data show that FNfn10-3JCL14 is a specific, high-affinity alphavbeta3-binding protein that can inhibit alphavbeta3-dependent cellular processes similar to an anti-alphavbeta3 monoclonal antibody. These properties, combined with the small, monomeric, cysteine-free and highly stable structure of FNfn10-3JCLI4, may make this protein useful in future applications involving detection and targeting of alphavbeta3-positive cells.     

Linker effects on biological properties of 111In-labeled DTPA conjugates of a cyclic RGDfK dimer

In this report, we present in vitro and in vivo evaluation of three 111 In-labeled DTPA conjugates of a cyclic RGDfK dimer: DTPA-Bn-SU016 (SU016 = E[c(RGDfK)] 2; DTPA-Bn = 2-( p-isothioureidobenzyl)diethylenetriaminepentaacetic acid), DTPA-Bn-E-SU016 ( E = glutamic acid) and DTPA-Bn-Cys-SU016 (Cys = cysteic acid). The integrin alpha vbeta 3 binding affinities of SU016, DTPA-Bn-SU016, DTPA-Bn-E-SU016, and DTPA-Bn-Cys-SU016 were determined to be 5.0 +/- 0.7 nM, 7.9 +/- 0.6 nM, 5.8 +/- 0.6 nM, and 6.9 +/- 0.9 nM, respectively, against 125 I-c(RGDyK) in binding to integrin alpha vbeta3, suggesting that E or Cys residue has little effect on the integrin alpha vbeta3 affinity of E[c(RGDfK)] 2. It was also found that the 111 In-labeling efficiency of DTPA-Bn-SU016 and DTPA-Bn-E-SU016 is 3-5 times better than that of DOTA analogues due to fast chelation kinetics and high-yield 111 In-labeling under mild conditions (e.g., room temperature). Biodistribution studies were performed using BALB/c nude mice bearing U87MG human glioma xenografts. 111 In-DTPA-Bn-SU016, 111 In-DTPA-Bn-E-SU016, and 111 In-DTPA-Bn-Cys-SU016 all displayed rapid blood clearance. Their tumor uptake was comparable between 0.5 and 4 h postinjection (p.i.) within experimental error. 111 In-DTPA-Bn-E-SU016 had a significantly lower ( p < 0.01) kidney uptake than 111 In-DTPA-Bn-SU016 and 111 In-DTPA-Bn-Cys-SU016. The liver uptake of 111 In-DTPA-Bn-SU016 was 1.69 +/- 0.18% ID/g at 24 h p.i., while the liver uptake values of 111 In-DTPA-Bn-E-SU016 and 111 In-DTPA-Bn-Cys-SU016 were 0.55 +/- 0.11% ID/g and 0.79 +/- 0.15% ID/g at 24 h p.i., respectively. Among the three 111 In radiotracers evaluated in this study, 111 In-DTPA-Bn-E-SU016 has the lowest liver and kidney uptake and the best tumor/liver and tumor/kidney ratios. Results from metabolism studies indicated that there is little metabolism (<10%) for three 111 In radiotracers at 1 h p.i. Imaging data showed that tumors can be clearly visualized at 4 h p.i. with good contrast in the tumor-bearing mice administered with 111 In-DTPA-Bn-E-SU016. It is concluded that using a glutamic acid linker can significantly improve excretion kinetics of the 111 In-labeled E[c(RGDfK)] 2 from liver and kidneys.     

ADAM15 decreases integrin alphavbeta3/vitronectin-mediated ovarian cancer cell adhesion and motility in an RGD-dependent fashion

We have recently described that integrin alphavbeta3 upon interaction with its major extracellular matrix ligand vitronectin induces adhesion, motility, and proliferation of human ovarian cancer cells. Due to the important function of alphavbeta3 in cancer cell biology, it has been the effort of many scientific approaches to specifically target alphavbeta3-mediated cell adhesion and tumorbiological effects arising thereof by synthetic integrin antagonists. More recently, proteins of the ADAM family have been recognized as naturally occurring integrin ligands. Among those, human ADAM15 which encompasses the integrin binding RGD motif was shown to interact with integrin alphavbeta3. Thus, we investigated in human ovarian OV-MZ-6 cancer cells, expressing both ADAM15 and alphavbeta3, whether ADAM15 might affect alphavbeta3-mediated tumorbiological effects. We stably (over)expressed ADAM15 or its extracellular domain in OV-MZ-6 cells as well as respective ADAM15 mutants containing the tripeptide SGA instead of RGD. Cells (over)expressing ADAM15-RGD exhibited a significantly reduced alphavbeta3-mediated adhesion to vitronectin. Also, a significant time-dependent decline in numbers of cells cultivated on vitronectin was noticed. This effect was found to be rather due to impaired alphavbeta3-mediated cell adhesion than decreased cell proliferation rates, since de novo DNA synthesis was not significantly altered by elevated ADAM15 expression. Moreover, a substantially decreased random cellular motility was noticed as a function of ADAM15 encompassing an intact RGD motif. In conclusion, our results point to a physiological role of ADAM15 as a natural binding partner of integrin alphavbeta3 thereby loosening tumor cell adhesion to the underlying matrix and regulating tumor cell migration and invasion.     

Integrins alpha5beta1, alphavbeta1, and alphavbeta6 collaborate in squamous carcinoma cell spreading and migration on fibronectin

The expression of alphavbeta6 fibronectin/tenascin receptor integrin is induced in malignant transformation of oral epithelium. In this study, we demonstrate the contribution of alphavbeta6 as well as other fibronectin receptor integrins in squamous cell carcinoma (SCC) cell adhesion and migration. Of 11 SCC cell lines isolated from the head and neck area, 8 (73%) expressed alphavbeta6 integrin on the cell surface. Three cell lines were chosen for further functional experiments: 1 with relatively high, 1 with moderate, and 1 with minimal surface expression of alphavbeta6 integrin. In addition to alphavbeta6, all 3 cell lines expressed alpha5beta1 and alphavbeta1 fibronectin receptor integrins. Function-blocking experiments with inhibitory anti-integrin antibodies showed that all these three integrins were functional in SCC cell spreading on fibronectin. Integrin alphavbeta6, however, was not used as a primary but as an alternative fibronectin receptor by SCC cells, as the inhibitory anti-beta6 integrin antibody alone had no effect on spreading. In migration, however, alphavbeta6, alpha5beta1, and alphavbeta1 integrins were all used in cooperation. The presence of alphavbeta1 integrin in SCC cells is a novel finding as is its contribution to SCC cell migration. When one or two of these three receptors were blocked, the cells demonstrated an adaptive ability to remain migratory using integrins that were not targeted by antibodies. Utilization of a combination of receptors of different affinities may be beneficial for SCC cell migration versatility.     

MicroPET and autoradiographic imaging of breast cancer alpha v-integrin expression using 18F- and 64Cu-labeled RGD peptide

Cell adhesion molecules alphavbeta3 and alphavbeta5 play a pivotal role in tumor angiogenesis and metastasis. Antiangiogenic therapy by using small peptide antagonists of alphav-integrins slows tumor growth and prevents tumor spread. The ability to visualize and quantify integrin expression will enable selection of appropriate patients for clinical trials, following determination of treatment efficacy and development of new potent drugs. We have previously labeled cyclic RGD peptide c(RGDyK) with 125I and 18F and applied the radiotracers to both subcutaneous and orthotopic brain tumor models. Here we conjugated c(RGDyK) with 1,4,7,10-tetraaza-1,4,7,10-tetradodecane-N,N',N' ',N' "-tetraacetic acid (DOTA) and labeled the DOTA-RGD conjugate with 64Cu (t1/2) = 12.8 h, 19% beta+) in high radiochemical purity and specific activity. The tumor targeting ability and in vivo kinetics of 64Cu-DOTA-RGD was compared with [18F]FB-RGD and 125I-RGD in orthotopic MDA-MB-435 breast cancer model. All three radiotracers revealed fast blood clearance and high tumor-to-blood and tumor-to-muscle ratios. 125I-RGD had higher tumor uptake than the corresponding 18F and 64Cu analogues. [18F]FB-RGD indicated a fast tumor washout rate and an unfavorable hepatobiliary excretion pathway, resulting in significant activity accumulation in gallbladder and intestines. 64Cu-DOTA-RGD had prolonged tumor retention (1.44 +/- 0.09 %ID/g at 4 h postinjection) and persistent uptake in the liver. All three tracers revealed receptor specific tumor accumulation which were illustrated by effective blocking via coinjection with a blocking dose of c(RGDyK). Static microPET imaging and whole-body autoradiography showed strong contrast from the contralateral background. In conclusion, overall molecular charge and characteristics of radiolabels have profound effects on tumor accumulation and in vivo kinetics of radiolabeled RGD peptide. Further modification of the RGD peptide and optimization of the tracer for prolonged tumor uptake and improved in vivo kinetics are being explored.     

A mechanism for modulation of cellular responses to VEGF: activation of the integrins

Many similarities exist in the cellular responses elicited by VEGF and governed by integrins. Here, we identify a basis for these interrelationships: VEGF activates integrins. VEGF enhanced cell adhesion, migration, soluble ligand binding, and adenovirus gene transfer mediated by alphavbeta3 and also activated other integrins, alphavbeta5, alpha5beta1, and alpha2beta1, involved in angiogenesis. Certain tumor cells exhibited high spontaneous adhesion and migration, which were attributable to a VEGF-dependent autocrine/paracrine activation of integrins. This activation was mediated by the VEGFR2 receptor and regulated via phosphatidylinositol-3-kinase, Akt, and the PTEN signaling axis. Thus, integrin activation provides a mechanism for VEGF to induce a broad spectrum of cellular responses.     

Association between alphavbeta6 integrin expression, elevated p42/44 kDa MAPK, and plasminogen-dependent matrix degradation in ovarian cancer

Altered expression of alphav integrins plays a critical role in tumor growth, invasion, and metastasis. In this study, we show that normal human epithelial ovarian cell line, HOSE, and ovarian cancer cell lines, OVCA 429, OVCA 433, and OVHS-1, expressed alphav integrin and associated beta1, beta3, and beta5 subunits, but only ovarian cancer cell lines OVCA 429 and OVCA 433 expressed alphavbeta6 integrin. The expression of alphavbeta6 in OVCA 429 and OVCA 433 was far higher than alphavbeta3 and alphavbeta5 integrin and correlated with high p42/p44 mitogen activated protein kinase (MAPK) activity and high secretion of high molecular weight urokinase plasminogen activator (HMW-uPA), pro-metalloproteinase 2 and 9 (pro-MMP-9 and pro-MMP-2). In contrast to HOSE and OVHS 1, OVCA 433 and OVCA 429 exhibited approximately 2-fold more plasminogen-dependent [3H]-collagen type IV degradation. Plasminogen-dependent [3H]-collagen IV degradation was inhibited by inhibitor of uPA (amiloride) and MMP (phenanthroline) and by antibodies against uPA or MMP-9 or alphavbeta6 integrin, indicating the involvement of alphavbeta6 integrin, uPA and MMP-9 in the process. The alphavbeta6 correlated increase in HMW-uPA and pro-MMP secretion could be inhibited by tyrosine kinase inhibitor genistein or the MEK 1 inhibitor U0126, consistent with a role of active p42/44 MAPK in the elevation of uPA, MMP-9, and MMP-2 secretion. Under similar conditions, genistein and U0126 inhibited plasminogen-dependent [3H]-collagen type IV degradation. These data suggest that sustained elevation of p42/44 MAPK activity may be required for the co-expression of alphavbeta6 integrin, which in turn may regulate the malignant potential of ovarian cancer cells via proteolytic mechanisms.     

Near-infrared optical imaging of integrin alphavbeta3 in human tumor xenografts

In vivo optical imaging is potentially useful for evaluating the presence of tumor markers that are targets of molecular medicine. Here we report the synthesis and characterization of integrin alphavbeta3-targeted peptide cyclo(Lys-Arg-Gly-Asp-Phe) [c(KRGDf )] labeled with fluorescence dyes with wavelength spanning from the visible/near infrared (Cy5.5) to the true near infrared (IRDye800) for optical imaging. In vitro, the peptide-dye conjugates bound specifically to tumor cells expressing alphavbeta3. When administered intravenously into mice at a dose of 6 nmol /mouse, the conjugates accumulated in tumors expressing alphavbeta3. The tumor-to-background ratios for human KS1767 Kaposi's sarcoma in mice injected with Cy5.5-c(KRGDf ) and Cy5.5 were 5.5 and 1.5, respectively. Preinjection of c(KRGDf ) blocked the uptake of Cy5.5-c(KRGDf ) in tumors by 89%. In alphavbeta3-positive M21 and alphavbeta3-negative M21-L human melanoma, fluorescence intensity in the tumor of mice injected with IRDye800 - c(KRGDf ) was 2.3 and 1.3 times that in normal tissue, respectively. Dynamic imaging revealed that Cy5.5- c(KRGDf ) was rapidly taken up by KS1767 tumor immediately after bolus injection. The rate of its uptake in the tumor was reduced by preinjection of c(KRGDf ) in an interval time-dependent manner. Our data suggest that near-infrared fluorescence imaging may be applied to the detection of tumors expressing integrin alphavbeta3 and to the assessment of the optimal biological dose and schedule of targeted therapies.     

Specificity of the VP1 GH loop of Foot-and-Mouth Disease virus for alphav integrins

Foot-and-mouth disease virus (FMDV) can use a number of integrins as receptors to initiate infection. Attachment to the integrin is mediated by a highly conserved arginine-glycine-aspartic acid (RGD) tripeptide located on the GH loop of VP1. Other residues of this loop are also conserved and may contribute to integrin binding. In this study we have used a 17-mer peptide, whose sequence corresponds to the GH loop of VP1 of type O FMDV, as a competitor of integrin-mediated virus binding and infection. Alanine substitution through this peptide identified the leucines at the first and fourth positions following RGD (RGD+1 and RGD+4 sites) as key for inhibition of virus binding and infection mediated by alphavbeta6 or alphavbeta8 but not for inhibition of virus binding to alphavbeta3. We also show that FMDV peptides containing either methionine or arginine at the RGD+1 site, which reflects the natural sequence variation seen across the FMDV serotypes, are effective inhibitors for alphavbeta6. In contrast, although RGDM-containing peptides were effective for alphavbeta8, RGDR-containing peptides were not. These observations were confirmed by showing that a virus containing an RGDR motif uses alphavbeta8 less efficiently than alphavbeta6 as a receptor for infection. Finally, evidence is presented that shows alphavbeta3 to be a poor receptor for infection by type O FMDV. Taken together, our data suggest that the integrin binding loop of FMDV has most likely evolved for binding to alphavbeta6 with a higher affinity than to alphavbeta3 and alphavbeta8.     

Targeted inhibition of alphavbeta3 integrin with an RNA aptamer impairs endothelial cell growth and survival

Alphavbeta3 integrin is a crucial factor involved in a variety of physiological processes, such as cell growth and migration, tumor invasion and metastasis, angiogenesis, and wound healing. Alphavbeta3 integrin exerts its effect by regulating endothelial cell (EC) migration, proliferation, and survival. Inhibiting the function of alphavbeta3 integrin, therefore, represents a potential anti-cancer, anti-thrombotic, and anti-inflammatory strategy. In this study, we tested an RNA aptamer, Apt-alphavbeta3 that binds recombinant alphavbeta3 integrin, for its ability to bind endogenous alphavbeta3 integrin on the surface of cells in culture and to subsequently affect cellular response. Our data illustrate that Apt-alphavbeta3 binds alphavbeta3 integrin expressed on the surface of live HUVECs. This interaction significantly decreases both basal and PDGF-induced cell proliferation as well as inhibition of cell adhesion. Apt-alphavbeta3 can also reduce PDGF-stimulated tube formation and increase HUVEC apoptosis through inhibition of FAK phosphorylation pathway. Our results demonstrate that by binding to its target, Apt-alphavbeta3 can efficiently inhibit human EC proliferation and survival, resulting in reduced angiogenesis. It predicts that Apt-alphavbeta3 could become useful in both tumor imaging and the treatment of tumor growth, atherosclerosis, thrombosis, and inflammation.