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Novel design principles enable specific targeting of imaging and therapeutic agents to necrotic domains in breast tumors
- Liat Goldshaid1,
- Efrat Rubinstein1,
- Alexander Brandis1,
- Dadi Segal1,
- Noa Leshem1,
- Ori Brenner2,
- Vyacheslav Kalchenko2,
- Doron Eren3,
- Tamar Yecheskel3,
- Yoseph Salitra3,
- Yoram Salomon†4 and
- Avigdor Scherz†1Email author
DOI: 10.1186/bcr2579
© Goldshaid et al.; licensee BioMed Central Ltd. 2010
Received: 28 October 2009
Accepted: 24 May 2010
Published: 24 May 2010
Necrosis at the tumor center is a common feature of aggressive breast cancers and has been associated with poor prognosis. It is commonly identified by means of invasive histopathology, which often correlates with morbidity and potential tumor cell dissemination, and limits the reconstruction of the whole necrotic domain. In this study we hypothesized that non covalent association to serum albumin (SA) and covalent binding to ligands for tumor-abundant cell receptors should synergistically drive selective accumulation and prolonged retention of imaging and therapeutic agents in breast tumor necrotic domains enabling in vivo identification, imaging and possibly treatment of such tumors.
Cyclo-Arg-Gly-Asp-D-Phe-Lys (c(RGDfK)) were conjugated to bacteriochlorophyll-derivatives (Bchl-Ds), previously developed as photodynamic agents, fluorescent probes and metal chelators in our lab. The c(RGDfK) component drives ligation to αVβ3 integrin receptors over-expressed by tumor cells and neo-vessels, and the Bchl-D component associates to SA in a non-covalent manner. STL-6014, a c(RGDfK)-Bchl-D representative, was i.v. injected to CD-1, nude female mice bearing necrotic and non-necrotic human MDA-MB-231-RFP breast cancer tumors. The fluorescence signals of the Bchl-Ds and RFP were monitored over days after treatment, by quantitative whole body imaging and excised tumor/tissue samples derived thereof. Complementary experiments included competitive inhibition of STL-6014 uptake by free c(RGDfK), comparative pharmacokinetics of nonconjugated c(RGDfK) Bchl-D (STL-7012) and of two human serum albumin (HSA) conjugates: HSA-STL-7012 and HSA-STL-6014.
STL-6014 and STL-7012 formed complexes with HSA (HSA/STL-6014, HSA/STL-7012). STL-6014, HSA-STL-7012 and HSA-STL-6014, selectively accumulated at similar rates, in tumor viable regions over the first 8 h post administration. They then migrated into the necrotic tumor domain and presented tumor half lifetimes (T1/2) in the range of days where T1/2 for HSA-STL-6014 > STL-6014 > HSA-STL-7012. No accumulation of STL-7012 was observed. Pre-injection of c(RGDfK) excess, prevented the uptake of STL-6014 in the small, but not in the large tumors.
Non-covalent association to SA and covalent binding to c(RGDfK), synergistically enable the accumulation and prolonged retention of Bchl-Ds in the necrotic regions of tumors. These findings provide novel guidelines and strategy for imaging and treatment of necrotic tumors.
Upon outgrowth of their oxygen supply, emerging tumors develop hypoxic and eventually necrotic regions believed to be the direct result of chronic ischemia caused by vascular collapse when the rate of tumor cell growth exceeds that of angiogenesis [1]. Initially, necrotic areas within solid tumors constitute distinct morphologically identifiable regions that contain highly eosinophilic cells. Subsequently, this pattern is replaced by liquefaction necrosis, in which the breakdown of cellular structures can be observed [1]. Necrosis in invasive carcinoma of the breast has been correlated with the concomitant angiogenesis, development of high vascular density, and increased levels of focal macrophage infiltration driven by chemotactic factors [1–4].
Assessment of the degree of necrosis has been proposed to serve as a powerful prognostic marker for different cancer types [5–7]. Necrosis at the tumor center is a common feature of aggressive breast cancer and has been associated with poor prognosis. In particular, large necrotic volumes in ductal carcinoma in situ (DCIS) demonstrate tight correlation with comedo as opposed to non-comedo DCIS [8]. Necrosis in this tumor type was shown to be associated with hypoxia [4], where prolonged hypoxic conditions were found to increase the rates of mutations, tumor progression and angiogenesis, and to promote metastatic potential and pre-growth signaling pathways. In summary, the magnitude of necrosis in mammary ducts appears to positively correlate and possibly predict DCIS aggressiveness [9]. Necrotic and hypoxic conditions constitute major impediments to successful cancer therapy [10]. Therefore, increased detective resolution of hypoxic and necrotic tumors in vivo will enhance diagnostic procedures and better outline the tumor status for selection of appropriate treatment strategies [11–14].
Necrosis and hypoxia are commonly identified by means of invasive histopathology, which is often correlated with morbidity and potential tumor cell dissemination. Thus, in situ diagnostic techniques, such as magnetic resonance imaging (MRI) [15, 16], blood oxygenation level dependent-MRI [17], diffusion-weighted MRI [18] and positron emission tomography (PET) [19] can be advantageous. However, insufficient vascularization, linked with enhanced interstitial fluid pressure due to the lack of lymphatic drainage, interfere with the convectional uptake of small molecules used as therapeutic or contrast agents in such detection protocols. At the same time, enhanced tumor vascular permeability in these regions drives extravasation of macromolecules such as serum albumin (SA) from the circulation into the tumor tissue, while the poor lymphatic drainage fosters their retention within the tumor compartment [20, 21]. Consequently, the 'enhanced permeability and retention effect' (EPR), has been proposed as the basis for nonspecific targeting of drugs comprising large molecules to tumor tissue and has given rise to a new approach for tumor-targeting drug design based on macromolecular, micellar and lipidic particles (for a recent review see Maeda and colleagues [22]). For example, non-covalent association of styrene-maleic acid copolymer-conjugated neocarzinostatin (16 kDa) with albumin, provides about an 80 kDa complex that accumulates and exhibits retention in tumors of different origins [22]. Similarly, covalent conjugation of the therapeutic agents mitomycin C-bovine or indotricarbocyanine (ITTC) to SA, resulted in their delivery to and accumulation/retention in tumors and were even detected in the tumor necrotic domains [23]. Gadophrin-2A, a porphyrin-based necrosis avid contrast agents (NACA) used as an MRI contrast enhancing agent in necrotic tumor imaging, has been proposed to accumulate in the margins of HT29 human colon tumor necrotic zones, following delivery as a non-covalent complex with SA either as is or after dissociation in the tumor necrotic domain [24].
Recent studies showed better accumulation of NACA, which have been conjugated to large molecules that function as ligands to over-expressed tumor cell receptors. Thus, the ITTC accumulation in HT29 human colon tumors was improved after its covalent conjugation to transferrin in place of albumin, presumably because of the transferrin interactions with the cancer cell surface receptors [25]. However, such high molecular weight complexes may elicit an immune response that should prevent their frequent application as needed for imaging and therapeutic applications. Thus, despite the advances made in tumor-targeting techniques there is a clear need to recruit new imaging principles and to expedite the development of novel agents for such purposes.
In our quest for new imaging avenues, we postulated that small contrast and/or therapeutic agents designed to have the dual capacity of moderate association affinity to SA and high affinity to tumor-specific receptors, would allow for their prolonged accumulation in necrotic tumor domains. We assumed that the EPR effect would assist agent extravasation and retention in the tumor interstitium upon forming a complex with SA. Once in the tumor tissue, the modified agents will dissociate from the SA and preferentially bind to specific receptors, ensuring active agent accumulation and possibly migration to the necrotic domain by means of diffusion. Notably, the design principle of such non-covalent complexes with SA is profoundly different from the one underlying the make up of covalent SA complexes [26].
Herein, we examined the application of cyclic compounds containing the Arg-Gly-Asp (RGD) sequence as to impart tumor-specific ligands for targeting Bchl-Ds/SA to necrotic tumor domains. The RGD motif is recognized by many members of the integrin family [41]. More specifically, c(RGDfK), and similar compounds demonstrated significant affinity to αVβ3 integrin receptors that are abundant in several cancer cell lines and in the rapidly developing neo-vasculaturization near necrotic tumor domains. Hence, RGD-containing molecules have been suggested to provide new opportunities for selective tumor targeting of imaging and therapeutic agents [42]. One of the problems with such application is the short life-time of the small RGD complexes in the circulation (two to three minutes lifetimes for c(RGDfK) [43]. The covalent binding of cyclic RGD to macromolecules such as human SA (HSA), results in a better accumulation of the cyclic RGD compound in tumors probably because of the significantly enhanced life time of the RGD-HSA complex in the circulation compared with the free RGD [26] and the involvement of the aforementioned EPR effect. However, as noted above the resulted protein complex may be immunogenic and therefore of limited use in the clinical arena. Hence, mobilization of the therapeutic or contrast agents by reversible, non-covalent association in situ with circulating SA, as proposed in the present study, is expected to be advantageous.
Recently, we synthesized and tested the uptake by tumors of STL-6009, a c(RGDfK)-conjugated WST11 (Figure 1, [37, 44]). The compound selectively accumulated to significant concentrations (4 to 8 μM) in primary xenografts in mice. These concentrations mirrored the circulating Cmax values recorded for nonconjugated WST11 using inductively coupled plasma mass spectroscopy measurements. STL-6009 accumulation exhibited tight dependence on the presence of αVβ3 receptors in the respective tumor cell lines and on the extent of neovascularization in the xenograft model [37]. However, the weak fluorescence of STL-6009 due to the central palladium (Pd) atom [45], impaired the clarity of on-line fluorescence imaging, rendering its further development for imaging purposes futile. Thus, in the present study the central Pd atom was substituted by two protons yielding STL-6014 (Figure 1), a novel RGD-Bchl-D conjugate that emits 100-fold stronger fluorescence than its Pd-chelated counterpart. STL-6014 uptake was monitored in mice bearing MDA-MB-231-red fluorescence protein (RFP) breast tumors with and without central necrosis and compared with that of the following compounds: (1) STL-7012, an RGD-free analog; (2) HSA-STL-6014; and (3) HSA-STL-7012. The experimental findings combined with some literature data strongly support the above suggested hypothesis. Namely, non-covalent association to SA, and covalent binding to ligands of tumor-abundant cell receptors, synergistically drive selective accumulation and prolonged retention of agents in the necrotic domains of breast tumors, enabling the in vivo imaging of such necrotic tumors.
Materials and methods
Bchl-D preparation
Bchl-Ds including 31-oxo-15-methoxycarbonylmethyl-Rhodobacteriochlorin 131-(2-sulfoethyl)amide dipotassium salt (STL-7012, Figure 1a), and its palladium complex (WST11, Figure 1b), 31-oxo-15-methoxycarbonylmethyl-Rhodobacteriochlorin 131-(2-sulfoethyl)amide-173-c(RGDfK)amide potassium salt (STL-6014, Figure 1c), and its palladium complex (STL-6009, Figure 1c), were prepared as previously described [37, 38]. The Bchl-Ds were dissolved in 5% aqueous mannitol and adjusted to a pH of 7.2 to 7.4 with Tris HCl (10 mM tris(hydroxymethyl) aminomethane in 5% aqueous mannitol). The obtained solutions were filtered through 0.2 μm polytetrafluoroethylene (PTFE) filters (National Scientific, Rockwood Tennessee, USA). Concentrations were spectrophotometrically determined in methanol at 747 nm, using molar extinction coefficients of 1.2 × 105 M-1cm-1 for Pd-containing and 6.3 × 104 M-1cm-1 for Pd-free compounds. The solutions were stored in the dark at -20°C until use.
HSA-STL-6014 and HSA-STL-7012 preparation
c(RGDfK)-bacteriopheophorbide a (Bpheid) was synthesized via activation of Bpheid [38] with N-hydroxysuccinimide (NHS) and N,N-dicyclohexylcarbodiimide (DCC) in tetrahydrofuran, followed by purification of activated esters on silica column and then reaction with two-fold molar excess of c(RGDfK) in dimethylformamide (DMF) in the presence of triethylamine under argon atmosphere overnight (yield about 85%). Aminolysis of Bpheid-c(RGDfK) and Bpheid dissolved in chloroform-methanol mixture was induced with bis(3-aminopropyl)amine (yield >90%). The resulting Bchl-Ds were first purified using n-butanol-water extraction, and then by High-performance liquid chromatography (HPLC), using C18-column and acetonitrile-water gradient. HSA (90 mg) was activated with sulfoNHS and 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) (sulfoNHS:EDAC:HSA = 1000:500:1 molar ratio) in 50 mM phosphate buffer, pH 5.0. After 45 minutes, a sample of the reaction mixture (about 1 mL) was passed through PD-10 column (Sephadex G-25 M, GE Healthcare, Uppsala, Sweden) using 5 mL of the same buffer to recover the protein fraction (HSA-sNHS). Bchl-Ds (about 20 mg each) were dissolved in DMF (2 mL) and added to 2 mL HSA-sNHS, diluted with 4 mL of 50 mM phosphate buffer, pH 8.0, and 1 ml of 5% mannitol. Reaction mixtures were sonicated and stirred overnight under argon atmosphere, then evaporated to remove DMF, re-dissolved in water and passed again through PD-10 column for collection of Bchl-D-HSA-conjugates. To remove non-covalently bound Bchl-D, the products were evaporated and extracted several times with methanol. The extent of HSA conjugation was calculated from the molar ratios between the Bchl-D and HSA parts, determined spectrophotometrically and by Bradford assay, respectively. Approximately 10% of HSA was conjugated. Aliquots of conjugates equal to 0.7 nmol Bchl-D, were prepared for injections.
Cell culture
MDA-MB-231 breast cancer cells were obtained from the American Type Culture Collection (Manassas, VA, USA). MDA-MB-231-RFP were generated by stable transfection of the cell line with pDsRed-Monomer-Hyg-C1 (Clontech, Palo Alto, CA, USA), which carries the hygromycin resistance gene in which the DsRed-Monomer gene was replaced with pDsRed2 (from pDsRed2-N1 plasmid, Clontech, Palo Alto, CA, USA). Lipofectamine™ 2000 (Invitrogen, Carlsbad, California, USA) was used for the transfection process according to the manufacturer's protocol. Stably RFP-expressing cells were then cloned and maintained in RPMI 1640 medium supplemented with 1 mmol/L sodium pyruvate, 10% fetal calf serum, 250 μg/ml hygromycin, 0.06 mg/ml penicillin and 0.1 mg/ml streptomycin.
Association constants of Bchl-Ds to HSA
Where [SA]0 is the analytical concentration of the added SA and [(Bchl-D/SA)] is the concentration of the Bchl-D complex with SA calculated from the spectral changes.
Female, CD-1, nude mice (7 to 8 week old, about 25 g) were housed and handled with ad libitum access to food and water at the Core Animal Facility according to Institutional Animal Care and Use Guidelines. All experimental procedures were approved by the Institutional Animal Care and Use Committee at the Weizmann Institute of Science (Rehovot, Israel).
Mouse tumor model
Mammary fat pads (left, bottom nipple) of female mice were inoculated with harvested MDA-MB-231-RFP human breast cancer cells (4 × 106 in 100 μl saline). Tumors were classified as 'small' (≤100 mm3) at one to two weeks from time of cell injection, or 'large' after three to four weeks (necrotic tumors, 250 to 500 mm3). To avoid tumor burden, mice were sacrificed (cervical dislocation) when tumor size reached 10% of body weight or at 90 days post-implantation. External caliper measurements, length (L), width (W) and depth (D) were used to calculate in vivo tumor volume according to the formula: V = L/2 · W/2 · D/2 · π · 4/3 [46].
Whole-body fluorescence imaging
The in Vivo Optical Imaging System (IVIS®100/XFO-12, Xenogen Corp., Alameda, CA, USA) was used to acquire fluorescent images of the RFP-expressing tumors, as well as Bchl-D compounds associated with tissues and organs after their intravenous (i.v.) infusion to the tested animals. The field of view was 15 cm. RFP fluorescence, in units of photons/sec, was detected using 525/50 nm and 612/75 nm filter sets for excitation and emission, respectively, with one second integration time. NIR fluorescence of the different Bchl-Ds, in units of photons/sec, was detected using 680/30 nm and 847/75 nm filter sets for excitation and emission, respectively, at an integration time of five seconds. Background fluorescence for all quantitative analyses was calculated by recording the average fluorescence (photons/sec)/cm2 from three animals in three different areas around and within the tumor of untreated animals provided with a similar diet for three days before measurements.
Drug accumulation was determined following i.v. administration of molar equivalents of the Bchl-D moiety to the tail vein of mice fed with a chlorophyll-free, purified diet (Harlan Teklad, Harlan Laboratories, Indianapolis, IN, USA) for three days before administration (in order to reduce skin and food autofluorescence). Before imaging, mice were anesthetized by intraperitoneum injection of a 30 μl mixture of 85:15 ketamine:xylazine.
Xenogen Living Image Software (Xenogen Corp., Alameda, CA, USA) was used for sequential fluorescent image acquisition [see Additional file 1] and superimposition of photographic images of mice and color-coded fluorescent images. Statistical analysis was performed using Origin8.1 (OriginLab, Northampton, Massachusetts, USA) and SPSS15 (SPSS Inc, Chicago, Illinois, USA) softwares.
Biodistribution studies
Anaesthetized mice were i.v. injected with 15 mg/kg STL-6014 (n = 27) or 9 mg/kg STL-7012 (n = 15). At each indicated time point, three animals were sacrificed and blood, kidney, liver and tumor tissues were collected into pre-weighed vials and frozen at -20°C. Tissue samples were homogenized in methanol (100 mg tissue/ml) and extracted the next morning by centrifugation (13,000 × g five minutes) at room temperature. Supernatants were diluted in methanol by a factor of two and drug concentration was determined by fluorescence measurements (Varian - Cary Eclipse spectrofluorimeter, Palo Alto, California, USA) at 750 nm (peak values). Drug concentrations were interpolated from a fluorescence calibration curve, based on predetermined drug concentrations using absorption spectroscopy.
In vivo competition between STL-6014 and free c(RGDfK) in MDA-MB-231-RFP tumors
STL-6014 (140 nmol, 7.5 mg/kg) was i.v. injected to the tail vein of mice one hour after being pre-injected with an excess (8.5 μmol) of c(RGDfK) free peptide. Whole-body fluorescence images were recorded up to 24 hours post-STL-6014 injection (IVIS®, Xenogen Corp., Alameda, CA, USA) with the respective filter settings, described above.
Histology and in vitro calculations of tumor and necrosis volumes
Tumors were surgically removed from sacrificed mice, fixed in 3.7% formaldehyde, and embedded in paraffin blocks. Tumors were serially dissected to 4 μm slices with a separation of 0.5 mm between consecutive slices. Slices were stained with H&E under standard conditions and evaluated for pathological markers (E800 microscope equipped with a Nikon DXM1200 digital camera, magnification × 0.6, objective × 0.5, Nikon, Tokyo, Japan).
where n is the number of slices for the evaluated tumor.
Necrotic MDA-MB-231-RFP tumor model
Above 90% of the large tumors (n = 30, 250 mm3 ≤Tv ≤500 mm3), presented central necrosis that occupies about 50% of Tv. Five to ten percent of the large tumors (n = 2) presented smaller volume of necrosis (about 50 mm3, about 15% of Tv). Conversely, more than 90% of the small tumors (n = 20, Tv ≤ 100 mm3) presented almost no necrosis. In 10% or less of these tumors (n = 2) a central necrosis (Tv ≤ 15 mm3 ≤20% of Tv) is observed. Thus, central necrosis appears to correlate only with large tumors of MDA-MB-231-RFP cells in this mouse model.
STL-6014 uptake into orthotopic MDA-MB-231-RFP tumors
The tumor-targeting capabilities of RGD-conjugated Bchl-Ds, was tested by following the accumulation of i.v. injected STL-6014 in mice bearing large or small MDA-MB-231-RFP tumors.
Large tumors
Small tumors
Accumulation of STL-6014 in small tumors (n = 10) was assessed under the same experimental conditions but appeared markedly different (Figures 3c and 3d). Although peak fluorescence of the injected drug was evident within the tumor at six to eight hours after injection (Figure 3c, upper panel), its level relative to the surrounding tissue was rather small. Tumor fluorescence returned to background values within 48 h of drug injection (Figures 3c and 3d).
Quantitative assessment of the STL-6014 dynamic fluorescence in small and large tumors post i.v. administration
The temporal evolution and decay of STL-6014 fluorescence density ((number of photons at (810 to 870 nm))/(sec × cm2 tumor tissue)) was calculated and presented versus time for mice bearing large tumors (n = 9, Figure 3E) or small tumors (n = 10, Figure 3f). In both tumor types, STL-6014 concentrations peaked within six to eight hours of administration. This period of accumulation was then followed by a mono-exponential decay in the small tumors, with an average t1/2 value of 14.0 ± 1.5 (SD) hours (R2 ≥ 0.97, P < 0.001, n = 10). In contrast, clearance from the large, necrotic tumors exhibited a bi-exponential decay with average t11/2 values of 12.0 ± 3.5 hours during the first 24 hours and t21/2 of 210.0 ± 86.0 hours thereafter (R2 ≥ 0.98, P < 0.001, n = 9). No statistically significant difference was seen between the fluorescence decay constant in small tumors and the first decay constant of large tumors. Large tumors maintained significant concentrations of STL-6014 for prolonged periods, as evident by the tumor fluorescence still detectable at nine days, and similar to the levels detected on day seven post-administration [see Additional file 2]. Thus, the emerging situation is of a relatively rapid-in slow-out pattern of accumulation. These results were substantiated by detailed analysis of drug concentrations in the different organs/tissues, as described in the 'biodistribution and pharmacokinetics analysis' section below.
Notably, the RFP fluorescence from the tumor cells declined shortly after STL-6014 infusion (Figures 3a to 3d lower panels) and then increased again at rates that appeared to mirror the uptake (first hours) and clearance of the STL-6014 from the corresponding tumors. Hence, this phenomenon provides independent evidence for the differential accumulation and clearance patterns of STL-6014 in small and large tumors [see Additional file 4].
Selective STL-6014 accumulation in the necrotic domain of MDA-MB-231-RFP tumors
Cumulatively, these results illustrate progressive accumulation and selective retention of STL-6014 within the necrotic tumor domain after temporary lodging in the tumor viable region.
Biodistribution and pharmacokinetics of STL-6014
Is the c(RGDfK) moiety essential for the STL-6014 uptake by the tumor?
The role played by the Bchl-Ds association to SA in their uptake and prolonged accumulation in the MDA-MB-231-RFP tumors
Does the Bchl-D conjugation to the c(RGDfK) increases its binding affinity to SA and thereby tumor accumulation and retention?
Following the above findings, it may be suggested that stronger binding of STL-6014 to SA compared with RGD-free STL-7012 analogue, accounts for the increased accumulation of the former in tumors. Therefore, we set out to determine the association constants of the four Bchl-Ds presented in Figure 1 to HSA. The derived constants (Figure 1, in parenthesis) were found to be within the same order of magnitude. In fact, those for the RGD conjugates were four to six fold lower than the association constants of their RGD-free analogues, ruling out stronger SA association as the sole basis for STL-6014 retention in the tumor.
In summary, association to SA appears to be important for the uptake of Bchl-Ds to the necrotic tumor domain, but is not enhanced by the RGD moiety.
Does the contribution of the RGD moiety involve association with tumor-specific cell receptors?
As c(RGDfK) was found not to enhance the non-covalent association of the Bchl-Ds moiety (e.g. STL-7012) with circulating SA, emphasis was placed on defining the role of RGD in STL-6014 uptake by the tumor. The possible involvement of a specific RGD binding site such as αVβ3 integrin in the observed drug accumulation within tumors was next examined by designing a competition assay between STL-6014 and free c(RGDfK).
Small tumors
Large tumors
A similar competition experiment was performed for animals with large tumors. However, in this case the uptake and accumulation of STL-6014 was not affected by the pre-administration of free c(RGDfK) (data not shown).
Preferential targeting of contrast and therapeutic agents for detection, imaging and treatment of tumors is understood to bear great prospects for effective cancer management. Of particular significance is the early, in situ detection of necrotic tumors in patients with localized breast cancer. This is critical for the appropriate selection of a treatment approach.
This study introduces a new strategy for in vivo imaging of necrotic breast tumors, following the design of Bchl-D-based agents that associate non-covalently with circulating SA immediately upon i.v. administration, and contain ligands with acute affinity for tumor-specific receptors. The moderate association of Bchl-D with SA was postulated to assist extravasation and temporal retention of the Bchl-RGD molecules by the necrotic tumor domains, via the EPR effect, while their strong affinity to tumor-specific receptors would maintain prolonged residence in these domains.
To test this hypothesis, we introduced the STL-6014 conjugate (Fig. 1) to mammary tumor-bearing mice. Although STL-6014 moderately associates with SA (Ka = 1.2 × 105M-1, Figure 1), the c(RGDfK) moiety has been shown to strongly associate (Ka about 109M-1) with αVβ3 integrin receptors abundant in the tumor vasculature, the cell membrane of some cancer cells [42] and on macrophages that infiltrate to the tumor tissue. Hence, c(RGDfK) was expected to provide high tumor affinity to the tested RGD-Bchl-D conjugates during their circulation as complexes with SA.
By correlating the red fluorescence of the RFP-expressing cancer cells and the NIR fluorescence emitted by the different Bchl-Ds, we monitored the dynamics of their biodistribution, as well as their accumulation in the tumor, prolonged retention and compartmentalization in the tumor sub-domains. The presented data demonstrate that conjugation to c(RGDfK) and complexation with SA synergistically enhances the tumor uptake and retention of the tested RGD-Bchl molecules in tumor necrotic domains. The c(RGDfK) moiety was shown to be essential for tumor-specific drug accumulation, as evidenced by the lack of free unconjugated Bchl-D uptake (Figure 7) [44]. On the other hand the half life of STL-6014 (Figure 3) was much longer even in small tumors (about 12 hours) than that of free c(RGDfK) molecules in a similar animal model (minutes) [43]. This role is further demonstrated by the significantly slower clearance of the HSA-STL-6014 covalent conjugate compared with the HSA/STL-6014 complex (Figure 8c) and the HSA-7012 covalent conjugates (Figures 8b and 8c) compared with HSA/7012 complexes (Figures 6b and 7). The same observations were recently made after i.v. administration of STL-6009, a non-fluorescent c(RGDfK)-(Pd)Bchl-D [44].
As the c(RGDfK) moiety was found not to increase the Bchl-Ds association to the SA, its key role presumably reflects the hypothesized affinity to particular receptors in the tumor tissue. Intense affinity to αVβ3 integrins receptors has been previously proposed to account for the c(RGDfK)-based drug retention-enhancing effect. MDA-MB-231 human breast cancer cells, as well as tumor vasculature have been shown to express the αVβ3 receptor [47, 48]. In a recent study [44], we clearly correlated STL-6009 uptake by tumor xenografts of various cell types, with both their vascular density and the level of αVβ3 integrins expressed [44]. In the present study, the free circulating c(RGDfK) competition (Figure 9) provides additional evidence with regard to the role of RGD in active agent accumulation within tumors. However, the observation that administration of free c(RGDfK) molecules cannot compete with the uptake and accumulation of RGD-Bchl by large necrotic tumors is not in line with this simple explanation.
The differential response of large and small tumors to free c(RGDfK), complements the dramatic difference in STL-6014 accumulation patterns within the two tumor classes. Small tumors accumulated STL-6014 over the first four to eight hours post-drug injection, followed by its full clearance within less than two days. In contrast, large MDA-MB-231-RFP tumors containing necrotic regions accumulated STL-6014 at approximately the same rate, followed by a shift in STL-6014 localization to the central necrosis, where it reached peak concentration by 48 to 72 hours post-injection. This phase was followed by slow, bi-phasic clearance (Figures 3, 4 and 6a). The diverse clearance patterns were experimentally correlated with the viable and necrotic domains in the tumors, where the more rapid clearance was seen from viable regions. This conclusion was substantiated by findings that the fluorescence from 'large' tumors bearing small Nv, cleared significantly faster than from tumors of similar size but with larger Nv, namely smaller viable cells mass (Figure 4). The observation that the majority of 'small' tumors contained no or very small Nv (Figure 4) coincided with their monoexponential and fast fluorescence decay. Conversely, more than 90% of the large tumors featured large Nv, correlating with the bi-exponential clearance pattern (Figures 3, 4 and 6) [see Additional file 2].
In line with these observations, we propose the presence of two distinct Bchl-D-c(RGDfK) target sites. The first is suggested to comprise RGD-binding sites, presumably integrin receptors presented on neoendothelial cells in both small and large tumors. This site is further suggested to be easily accessible to both c(RGDfK)-Bchl-D and free c(RGDfK). The second target site correspond to the necrotic domains, involving integrin receptors presented by the regular tumor cells, eosinophylic necrotic tumor cells, and possibly infiltrating white blood cells. Interaction of RGD ligands with these integrins requires their extravasation and retention into the tumor interstitium. Such extravasation can be driven in the large necrotic tumors by the EPR effect that is relevant to the large c(RGDfK)-Bchl-D/SA complexes but not to the small, free c(RGDfK) molecules, which rapidly clear from the tumor [43]. Therefore, the free c(RGDfK) molecules cannot compete with c(RGDfK)-Bchl-D binding to cellular integrins in the large tumors. The prolonged retention of the c(RGDfK)-Bchl-D/SA within the large tumor shifts the equilibrium of the SA-bound Bchl-D-c(RGDfK) to a more stable, RGD-mediated interaction with the stationary integrin receptors through the RGD moiety. All together, the above findings indicate that the EPR effect may support mobilization of SA-associating therapeutic and imaging agents into the tumor necrotic domain. However, interactions with tumor-specific receptors are required for the prolonged retention and accumulation of these compounds within the tumor.
The sharp displacement of STL-6014 fluorescence from the viable to the necrotic domain within large tumors at increasing times after administration (Figures 4 and 5) [see also Additional file 5] presents a feature essential to diagnostic protocols in search of advanced tumors. This shift is believed to be driven by the diffusion pressure created by the imbalanced concentrations of c(RGDfK)-Bchl-D in the viable versus necrotic tumor domains. Neutrophils and possibly macrophages, which infiltrate the necrotic domain (Figure 2) [1] may participate in anchoring the migrating c(RGDfK)-Bchl-D molecules via αVβ3 integrin receptors, expressed at high levels [49, 50]. Alternatively, self-aggregation of c(RGDfK)-Bchl-D molecules within the semi-aqueous necrotic domain may occur due to their increased concentrations, shifting the equilibrium toward further accumulation. These possibilities are currently under investigation. Notably, Chen and colleagues [26], recently showed that covalent binding of specific cyclic RGD molecules to HSA enhances their in vivo accumulation in tumors compared with the HSA-free analogues. They investigators related this phenomenon to the increased circulating t1/2 of the covalent conjugates (21 minutes vs. two to three minutes).
The reproducible results presented in this study have been further supported by analysis of STL-6009 and STL-6014 behavior in the same MDA-MB-231-RFP tumors and in human MLS ovarian and renal cell carcinoma tumors, respectively, at various stages of necrotic development (data not shown).
Cumulatively, the presented data demonstrates selective accumulation and prolonged retention of c(RGDfK)-Bchl-D within the necrotic tumor domain in MDA-MB-231-RFP bearing mice. A clear reliance on the synergistic effect of both the RGD moiety and the agent's association to SA was illustrated.
Although the experiments described here utilized fluorescence for the selective imaging of necrotic tumors, they are highly relevant to the clinical setting. First, the orthotopic engraftment of human breast cancer to the mouse mammary pad offers an environment resembling human breast tumors. In addition, the central necrosis described here, reflects the natural development of breast tumors in humans. Furthermore, the RGD-mediated accumulation characterized in the present study is of relevance to human cancerous environments, where αVβ3 integrin expression is typically associated with neovessels, tumor cells and macrophages abundant in the necrotic tumor domain [47].
The described RGD/SA-mediated homing and prolonged retention of fluorescent Bchl-Ds in the tumor necrotic domain may enable early detection of tumor growth and foster prognosis and the development of novel modes of treatment, including drug-targeted photodynamic therapy. As previously reported, RGD-SA contrast agents [26] are likely to be immunogenic and/or toxic in the clinical setting. The strategy suggested in the present manuscript is not only simpler, but more versatile and expected to present minimal toxicity. The relatively rapid clearance of STL-6014 and other RGD-Bchl-Ds from the liver and spleen is of utmost importance in clinical application of developed drugs. Clearly, the use of STL-6014 in the clinical setting is limited by the optical technologies required for in-depth 3 D tumor detection. However, optical tomography of the breast can be useful for this purpose. Moreover, 64Cu and Mn Bchl-RGD conjugates have been recently prepared in our laboratory [37] and using inductively coupled plasma mass spectroscopy showed to undergo similar patterns of accumulation and retention in tumors as STL-6014. These new drugs are expected to open the way for in vivo imaging of deeper necrotic tumors, both in the preclinical and clinical settings using PET scan and MRI spectroscopies, respectively.
- Bchl-D:
bacteriochlorophyll derivative
- Bpheid:
bacteriopheophorbide a
- c(RGDfK):
cyclic arginine-glycine-aspartic acid-(D)phenylalanine-lysine
ductal carcinoma in situ
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
enhanced permeability and retention
hematoxylin and eosin
human serum albumin
- i.v.:
magnetic resonance imaging
- Na:
necrotic area
necrosis avid contrast agent
near infrared
- Nv:
necrosis volume
positron emission tomography
red fluorescence protein
arginine-glycine-aspartic acid
serum albumin
- Ta:
tumor area
- Tv:
tumor volume.
The authors would like to thank Dr. Cathy Melamed Bessudo for her assistance in preparation of the transfected cells, Ran Afik for his help in the biodistribution assays, Dr. Iddo Pinkas for helping with the statistical analysis and decay curves, Tamara Berkutzki for performing the histological evaluation and Lasley Braff for her secretarial assistance. A.S and Y.S. are the incumbents of the Robert and Yaddele Sklare Professorial Chair in Biochemistry and the Tillie and Charles Lubin Professorial Chair in Biochemical Endocrinology, respectively.
This study was supported in part by grants from Steba Biotech, France and the Susan Komen Foundation for the Cure, Grant #BCTR0707388. None of the funding organizations was involved in the study design; in the collection, analysis, and interpretation of data; in the writing of the manuscript; and in the decision to submit the manuscript for publication.
Authors' Affiliations
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- Tanaka T, Shiramoto S, Miyashita M, Fujishima Y, Kaneo Y: Tumor targeting based on the effect of enhanced permeability and retention (EPR) and the mechanism of receptor-mediated endocytosis (RME). Int J Pharm. 2004, 277: 39-61. 10.1016/j.ijpharm.2003.09.050.View ArticlePubMedGoogle Scholar
- Hofmann B, Bogdanov A, Marecos E, Ebert W, Semmler W, Weissleder R: Mechanism of gadophrin-2 accumulation in tumor necrosis. J Magn Reson Imaging. 1999, 9: 336-341. 10.1002/(SICI)1522-2586(199902)9:2<336::AID-JMRI28>3.0.CO;2-3.View ArticlePubMedGoogle Scholar
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This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


text:
Fruit Filling
|Dried peaches/Apricots
|1⁄2 Pound
|1 1⁄4 Cup (20 tbs)
|1⁄4 Teaspoon
|Ground cinnamon
|1 Teaspoon
|Ground nutmeg
|1⁄2 Teaspoon
|1⁄2 Cup (8 tbs), soaked in warm water
1 Soak peaches or apricots in hot water to cover until soft, about 30 minutes.
2 Simmer peaches in a small quantity of water over medium high heat until they are very soft. Drain fruit and cool, then mash or puree in an electric blender.
3 Add remaining ingredients and mix well. Use to fill empanadas.
Recipe Summary
Rate It
Nutrition Rank
Nutrition Facts
Serving size: Complete recipe
Calories 1804 Calories from Fat 26
% Daily Value*
Total Fat 3 g4.8%
Saturated Fat 0.9 g4.5%
Trans Fat 0 g
Cholesterol 0 mg
Sodium 511.2 mg21.3%
Total Carbohydrates 466 g155.2%
Dietary Fiber 25.1 g100.4%
Sugars 398.5 g
Protein 11 g22.6%
Vitamin A 98.4% Vitamin C 22%
Calcium 16.6% Iron 63.4%
*Based on a 2000 Calorie diet

subset: commoncrawl


text:
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text: How To Make Your Own PETA Friendly Tanooki SuitBrian Crecente11/16/11 1:00pm75EditPromoteGo to permalink Don't buy Ashcraft's explanation about why PETA is completely missing the point in its attack on Mario's Tanooki suit? Here's an animal friendly way to get your own temporary fur coat. Be warned, it involves rotting food, duct tape and probably just a smidge of leptospirosis.


text:
Voting starts in March for the Drupal Association Board election.
This is opening the issue for this version that corresponds to the issue that was already patched for the 1.x branches.
I just manually applied the patch that was written for the 6.x-1.x branch to this (6.x-2.0-beta1) and it seems to fix the problem.
Specifically, the issue I was encountering was that the dropdown selection was not appearing for the menu mini panel.
Comment #1DamienMcKenna CreditAttribution: DamienMcKenna commented
|Clobbers $form array
|» Release new D6 beta
The fix is already in the 6.x-2.x branch, the module just needs a new beta release.
Comment #2John Franklin CreditAttribution: John Franklin commented
A new beta (or even a 2.0 final) would be very welcome.
Comment #3joelpittet CreditAttribution: joelpittet commented
Closing to triage the issue queue, there will be no further menu_attributes releases for D6


text:
Blood flowing down like the black hair on her head
Tears falling on the grass like rain
Bruised cheek; cut hands; ripped jeans; torn shirt
But there was the only thing to do to pay her way
She always had a smile carved on her face
Though she had a secret that she had to hide
She told no one; just kept it to herself;
And kept it deep inside.
But year after year after year went by
It became too much and she started to cry
Behind locked bathroom doors with her head in her hands
She prayed for an escape. She prayed to die.
It didn't take long for the truth began to leak
People saw the red marks on the base of her neck
They began to talk and word got out
No surprise that her life got wrecked.
She got into fights and had to leave her school
Some say her parents knew but they didn't want to hear.
Their baby girl selling herself to dangerous men
Is truly the thing that all parents fear.
Overcome, one night she climbed the old oak tree,
With 3 yards of rope in her hand.
She tied the noose and slipped it around her neck.
She fell towards the ground but she didn't land.
© Copyright 2017 The Ragamufin. All rights reserved.
Status: Finished
Genre: Song Lyrics
powerful and though dark, brilliant!
Oh my gosh, THIS IS PERFECT! I love this like so much. argh! so well written
Short Story / Romance
Short Story / Romance
Short Story / Fantasy
Paste the link to picture in the entry below:
Paste the link to Youtube video in the following entry:
Cannot annotate a non-flat selection. Make sure your selection starts and ends within the same node.
An annotation cannot contain another annotation.
There was an error uploading your file.


text:
The Dark Side of Love: The Positive Role of Our Negative Feelings-- Anger, Jealousy, and Hate
G.P. Putnam's Sons, 1994 - Psychology - 306 pages
A guide for using negative feelings in a constructive manner draws on case histories and ancient myths to debunk simplistic theories about love and to show how the acceptance of constructive expressions of anger, jealousy, and competition can enhance intimacy.
What people are saying - Write a review
The dark side of love: the positive role of our negative feelings-- anger, jealousy, and hateUser Review - Not Available - Book Verdict
Love has a dark side, claims practicing psychoanalyst Goldberg, whose main thesis is that all relationships evoke mixed feelings. At times everyone hates his or her spouse, child, or parent, and ... Read full review
Review: Dark Side of LoveUser Review - Konstantina - Goodreads
great book Read full review
The Myths of Love
Loving Love and Hating Hate
11 other sections not shown
Other editions - View all
The Dark Side of Love: The Positive Role of Negative Feelings
Jane G. Goldberg
No preview available - 1993
Common terms and phrases
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text:
Afghan policewoman kills U.S. civilian
KABUL A female police sergeant shot and killed a U.S. civilian adviser at police headquarters in Kabul on Monday, Afghan police said, breaking a relative lull in the so-called insider killings that have strained the relationship between Americans and Afghans here.
The victim was identified as Joseph Griffin, 49, of Mansfield, Ga., who had worked for DynCorp International as a police trainer since July 2011, according to a DynCorp spokeswoman, Ashley Burke.
Afghan officials identified the suspect as Nargis, 33, a sergeant in the national police force who worked in the Interior Ministry's legal and gender equality department, and whose husband is also a police officer.
A person at Kabul police headquarters said the attacker shot the U.S. adviser in the head at close range with a pistol and then was arrested by other Afghan officers. Police said she was distraught and that they did not believe the attack was terror-related.
Sixty-one U.S. and other coalition members have been killed by insider shootings this year, not including Monday's, compared with 35 the year before.
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text:
Eggplant With Yogurt
|1 , finely chopped
|2 Tablespoon
|Ripe tomatoes
|4 , diced
|1 Tablespoon
|Fresh ginger root/1/2 teaspoon ginger powder
|1 Teaspoon, chopped
|Vegetable salt
|1 1⁄2 Teaspoon
|Black pepper
|1⁄4 Teaspoon
|1 Large, cut in slices 3/4 inch thick
|Vegetable oil
|1⁄2 Cup (8 tbs)
|Vegetable salt
|1 Teaspoon
|1⁄2 Teaspoon
|Yogurt/Sour cream
|1 Cup (16 tbs)
|2 Tablespoon, finely chopped
Saute onion in margarine until golden brown.
Add tomatoes, and season with sugar, ginger, vegetable salt, and pepper.
Simmer, uncovered, until sauce thickens in about 10 minutes.
Dip eggplant slices in oil, coating each side well.
Arrange eggplant in a single layer in an electric skillet, or regular skillet, and simmer over a low heat for 30 minutes or until tender.
Turn eggplant occasionally so it's nicely browned on both sides.
Season with salt & pepper.
Place eggplant slices on a serving dish and spoon yogurt or sour cream over the top of each slice.
Top with a few tablespoons of tomato sauce in the center of each slice, leaving a border of yogurt or sour cream.
Garnish with parsley.
Rate It
Nutrition Rank
Nutrition Facts
Serving size: Complete recipe
Calories 1656 Calories from Fat 1271
% Daily Value*
Total Fat 144 g221.2%
Saturated Fat 23.7 g118.6%
Trans Fat 0 g
Cholesterol 29.4 mg9.8%
Sodium 4080.5 mg170%
Total Carbohydrates 88 g29.3%
Dietary Fiber 25.3 g101.2%
Sugars 55.5 g
Protein 20 g39.1%
Vitamin A 161.1% Vitamin C 207.1%
Calcium 44.4% Iron 28.2%
*Based on a 2000 Calorie diet


text:
Candy Memories
I remember as a kid in the late 60's early 70's I would go to Walgreen's drug store with my grandmother and my brother. My grandmother would always let us pick out whatever candy we wanted. My brother and I would always pick out the rock candy on a string that they sold there at the store. I'm glad to see that someone still sells it and I think I will start to get it for my grandchildren. Sue from Wisconsin
I admit to being a fiend for sugar. But sugar by itself was always too assertively sweet and it's sand-like consistency left much to be desired. Enter Rock Candy! The cloying sweetness had been tempered by some magical process and the crystals were large and smooth. The mouth-feel as it melted on my tongue was phenomenal! Each Saturday I would purchase a string or two from my favorite candy store, lodge it in the side of my cheek and suck contentedly til it bathed my mouth in pure Heavenly bliss! Memories are made of this! Lita from Arizona
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Posted by: CINDY
White Rock Candy
My candy arrived perfect..as described and the best service of any placed I have ordered from..plan to order again soon.
Quality Value Price
Posted by: Diana Blase
Purest form of candy
This is the first candy I had as a child, so it's kind of nostalgic, but I still love it! It's nice to just get something pure without a lot of ingredients, too. This is a good quality product, not too many "crumbs" in the box, either.
Quality Value Price


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Whole Business Securitization in Emerging Markets
Claire A. Hill
University of Minnesota, Twin Cities - School of Law
Duke Journal of Comparative and International Law, Vol. 12, No. 2, 2002
This short article, written for a symposium on securitization in emerging markets, discusses whole business securitization. Whole business securitization is a transaction structure used in countries with creditor-friendly bankruptcy regimes more friendly than the United States's regime. Whole business securitization specifies the manner in which a firm's business will be structured if it becomes bankrupt; the structuring expenditure is worthwhile in a regime where secured creditors' rights are fully honored. The article considers the extent to which the transaction structure will be useful in emerging markets countries. After the first transaction, in Malaysia, the structure is sure to be used again. But, not surprisingly, the structure is no panacea for the difficulties emerging markets firms encounter in raising capital.
Number of Pages in PDF File: 6
Keywords: bankruptcy, securitization, whole business securitization, creditors' rights, transaction structures, emerging markets
JEL Classification: F0, F3, G0
Date posted: March 11, 2003
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Goodell has suspended Roethlisberger for the first six games of the season but when he did, he said he could reduce the suspension to four games if the quarterback followed a righteous path. Goodell has since praised Roethlisberger for going "above and beyond" that path he set out for him.
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Manage and maintain international dating website
This project received 20 bids from talented freelancers with an average bid price of $6850 USD.Get free quotes for a project like this
Project BudgetN/A
Total Bids20
Project Description
I am looking for a partner to help develop the website [url removed, login to view]
The site is ready and its a great concept. People need to introduce themselves with a video which improves the experience of online dating as you get a much better idea of someone on a video (instead of a photo).
The website has some great features like an interview mode where a voice asks questions in order to make it easier to make a video.
The name is great and can be used anywhere in the world, and is trademark protected!
The partner will need to manage the site, which means:
- approving new videos
- maintaining and developing the software if needed
- work on SEO
- find ways to promote the site in creative (non expensive ways)
Ideally I would like to find someone who is willing to invest his time and belives in the project and helps to make it a succes. In return you will earn significant shares in our company. When succesfull this website can generate 100 of millions turnover, even only succesfull in 1 small country it can create some millions. But ofcourse this will not happen overnight, it will take energy and good work.
Please let me know if you are interested. Again I like to emphasize I am not looking at people who like to be paid upfront, but are willing to partner up and share the business results. The opportunity is huge!
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text: Even if you're not staying at the Kahala Hotel & Resort, it's worthwhile to park and walk around the grounds, filled with a lagoon, waterfalls, and flower gardens. A pair of dolphins live in the lagoon, and feeding time is a sight to behold. Kids will delight as the dolphins perform amazing antics in expectation of a lunch reward. Feeding times are 11:00 A.M. and 2:00and 4:00 P.M., and the show is free .Interesting The hotel that this is at was amazing! However, it was very strange when I showed up and told them I read a review that said they have a show here, they said there is no show but I did hvae the feeding times correct. We basically watched the Dolphins swim with the guests who paid for an incounter. My one year old slept the entire time by my husband and I enjoyed the show.


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fund something to provide money for something, usually something official a dance festival funded by the Arts Council The museum is privately funded. a government-funded programme See related entries: Running a business Word Origin mid 17th cent.: from Latin fundus
verbjump to other results
BrE BrE//fʌnd//; NAmE NAmE//fʌnd//Verb Forms present simple I / you / we / they fund
BrE BrE//fʌnd//; NAmE NAmE//fʌnd//he / she / it funds
BrE BrE//fʌndz//; NAmE NAmE//fʌndz//past simple funded
BrE BrE//ˈfʌndɪd//; NAmE NAmE//ˈfʌndɪd//past participle funded
BrE BrE//ˈfʌndɪd//; NAmE NAmE//ˈfʌndɪd//-ing form funding
BrE BrE//ˈfʌndɪŋ//; NAmE NAmE//ˈfʌndɪŋ//Running a business
Check pronunciation: fund


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Sprawl out on this spacious sectional sofa and show off your impeccable style to your friends. You'll be happy to entertain guests at your house with this sectional sofa's relaxed and stylish look and the flawless complement of contrasting toss pillows. But beauty isn't everything - this sofa also has reliable quality with heavy gauge steel seat springs, an expertly joined frame, and channeled fiber back cushions to maintain perfect support and structure.
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Heat shock protein 70 (HSP70) is a member of a highly conserved superfamily of intracellular chaperones called stress proteins that can activate innate and adaptive immune responses. We evaluated the effect of a fusion DNA vaccine that encoded mycobacterial HSP70 and MPT51, a major secreted protein of Mycobacterium tuberculosis. Spleen cells from mice immunized with fusion DNA of full-length HSP70 and MPT51 produced a higher amount of interferon-γ (IFN-γ) in response to the CD4+, but not the CD8+ T-cell epitope peptide on MPT51 than those from mice immunized with MPT51 DNA. Furthermore, because HSP70 comprises the N-terminal ATPase domain and the C-terminal peptide-binding domain, we attempted to identify the domain responsible for its enhancing effect. The fusion DNA vaccine that encoded the C-terminal domain of HSP70 and MPT51 induced a higher MPT51-specific IFN-γ production by CD4+ T cells than the vaccine that encoded MPT51 alone, whereas that with the N-terminal domain did not. Similar results were obtained by immunization with the fusion proteins. These results suggest that the DNA vaccine that encodes a chimeric antigen molecule fused with mycobacterial HSP70, especially with its C-terminal domain, can induce a stronger antigen-specific T-helper cell type 1 response than antigen DNA alone.
Tuberculosis is a major cause of death worldwide. There were an estimated 9.4 million incident and 11.1 million prevalent cases of tuberculosis, and 1.8 million people died of tuberculosis, in 2008 (World Health Organization, 2009). Mycobacterium bovis Bacillus Calmette–Guerin (BCG) is the only available vaccine against tuberculosis, but it has been reported to show variable protective efficacy (Colditz et al., 1994). Although BCG can protect against tuberculosis in childhood, its efficacy in adults varies, especially in endemic countries (Sterne et al., 1998; Kaufmann, 2004; Andersen, 2007). It is also known to represent a risk for immunocompromised patients. In addition, the emergence of multidrug-resistant strains of Mycobacterium tuberculosis has lent urgency to the search for novel therapeutic agents and the development of more effective vaccines capable of protecting adults (Kaufmann, 2000).
MPT51 is a major secreted protein of M. tuberculosis, which has a molecular weight of 27 kDa and a primary structure that is similar to the components of the antigen 85 complex (Nagai et al., 1991; Ohara et al., 1995, 1997). We demonstrated previously that MPT51 induces T-cell-mediated immune responses and protective immunity against challenge with M. tuberculosis in mice (Miki et al., 2004), and have identified one CD8+ and two CD4+ T-cell epitopes that are presented by H2-Dd and H2-Ab, respectively (Suzuki et al., 2004). MPT51 is recognized by T cells from patients with active tuberculosis and is also considered to be immunogenic in humans (de Araujo-Filho et al., 2008).
Heat shock protein70 (HSP70) is a member of a highly conserved superfamily of intracellular chaperones called stress proteins (Gething et al., 1995). In addition, HSP70 appears to play important roles in the innate and adaptive immune responses, such as receptor-mediated antigen internalization by sentinel antigen-presenting cells (APCs), stimulation of production of various cytokines, and maturation of dendritic cells (DCs) (Suto & Srivastava, 1995; Basu et al., 2000; Binder et al., 2000; Castellino et al., 2000; Singh-Jasuja et al., 2000). Collectively, the ability of HSP70 to bind antigenic peptides and deliver them to APCs profoundly contributes to the generation of antigen-specific T-cell responses.
In this study, we compared the effect of a DNA vaccine that encoded a fusion protein that consisted of MPT51, a protective antigen against M. tuberculosis (Miki et al., 2004), and M. tuberculosis HSP70 on the induction of MPT51-specific T-cell responses, with that of MPT51 DNA alone. After showing superior MPT51-specific T-cell induction by the fusion molecule, we attempted to establish the domain of HSP70 that was responsible for this enhancing effect.
Materials and methods
Construction of fusion genes for DNA vaccines
Mycobacterium tuberculosis HSP70 gene was cloned by PCR from genomic DNA of the H37Rv strain. The primers used for PCR were as follows:
HSP70F (full-length HSP70: residues 1–625)
5′-TATGAATTCACCATGGCTCGTGCGGTCGGG-3′ (forward)
5′-AATGGTACCCTTGGCCTCCCGGCCGT-3′ (reverse)
HSP70N (N-terminal domain of HSP70: residues 1–360)
5′-TATGAATTCACCATGGCTCGTGCGGTCGGG-3′ (forward)
5′-AATGGTACCCACCTCGCCCTTGAGGA-3′ (reverse)
HSP70C (C-terminal domain of HSP70: residues 354–625)
5′-TATGAATTCACCATGGCGTCCTCAAGGGC-3′ (forward)
5′-AATGGTACCCTTGGCCTCCCGGCCGT-3′ (reverse)
Forward and reverse primers contained EcoRI and KpnI restriction sites (underlined), respectively. PCR products were purified and cloned into the EcoRI/KpnI sites of the pCI vector (Promega, Madison, WI) that contained MPT51 DNA at the XbaI site (Suzuki et al., 2004). A nucleotide sequence for a 12-amino-acid (GGGSGGGSGGGS) linker was then inserted into the KpnI sites of pCI vectors that contained chimeric MPT51 DNA. The chimeric DNA constructs thus prepared were designated HSP70F-MPT51, HSP70N-MPT51, and HSP70C-MPT51. As a control, HSP70F was amplified by PCR using a primer set as follows: 5′-TATGAATTCACCATGGCTCGTGCGGTCGGG-3′ (forward, an underline indicates the EcoRI site) and 5′-AATGGTACCTCACTTGGCCTCCCGGCCGT-3′ (reverse, an underline indicates the KpnI site). The PCR product was purified and cloned into the EcoRI/KpnI sites of the pCI vector. The pCI plasmids containing these constructs were propagated in Escherichia coli strain XL1-Blue (Stratagene, La Jolla, CA) and purified by EndoFree Plasmid Maxi Kit (Qiagen GmbH, Hilden, Germany). The concentration of endotoxin was tested using the Endospecy ES-24S kit and the Toxicolor DIA kit (both from Seikagaku Biobusiness Corporation, Tokyo, Japan), and all plasmid solutions were found to contain <0.1 EU endotoxin µg−1 DNA.
Preparation of fusion proteins
For protein preparation, MPT51, HSP70F-MPT51, HSP70N-MPT51, and HSP70C-MPT51 were 6 × histidine-tagged by PCR using the corresponding forward primers listed above and a reverse primer (CAAGCTTTTAATGATGATGATGATGATGGCGGATCGCACCGACGATAT) containing nucleotide sequences for the extreme C-terminal of MPT51, the histidine-tag (double underlined), and the HindIII site (underlined). The PCR products were purified and cloned into the EcoRI/HindIII sites of the pRSET expression vector (Invitrogen, Carlsbad, CA). Escherichia coli JM109 competent cells were transformed with the expression vectors, and proteins were induced according to the manufacturer's instructions. Proteins were extracted and purified by Ni2+-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen GmbH) in the presence of 7 M urea, according to the manufacturer's instructions. Purified proteins were refolded by decreasing the concentration of urea while immobilized on the Ni-NTA matrix and then eluted by phosphate-buffered saline (PBS) (pH 7.4) containing 250 mM imidazole. The buffer was finally exchanged with PBS (pH 7.4) using a PD-10 desalting column (GE Healthcare, Uppsala, Sweden). The purity of recombinant proteins was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of endotoxin was tested using the Endospecy ES-24S kit and the Toxicolor DIA kit, and recombinant protein solutions contained <10 EU endotoxin mg−1 protein.
Preparation of anti-MPT51 monoclonal antibody (mAbs)
Anti-MPT51 mAbs were prepared as described previously (Harlow & Lane, 1988). Briefly, purified recombinant MPT51 protein (50 µg per mouse) was mixed with the RIBI Adjuvant System (Corixa, Hamilton, MT) and injected subcutaneously into BALB/c mice three times at 3-week intervals. Spleen cells from immunized mice were fused with SP2/0, and stably hybridized cells were selected using HAT medium (Invitrogen). Hybridomas that produced anti-MPT51 mAbs were screened by enzyme-linked immunosorbent assay (ELISA), and two clones (2B11F5 and 2D9H2) were established by limiting dilution. Both clones produced anti-MPT51 mAbs of the IgG1 isotype, as determined using a Mouse Immunoglobulin Isotype Kit (BD Biosciences Pharmingen, San Diego, CA).
Western blotting detection of chimeric MPT51 proteins
HEK293T cells were transfected with pCI plasmids that encoded MPT51 DNA of various forms using FuGENE 6 (Roche Diagnostics GmbH, Mannheim, Germany). Cells were collected 24 h after transfection and lysed in the radio-immunoprecipitation assay buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS), and Western blotting was performed with mouse anti-MPT51 mAb (from 2B11F5), HRP-conjugated goat anti-mouse immunoglobulins (Zymed Laboratories/Invitrogen), and West Pico Chemiluminescent Substrate (Pierce, Rockford, IL).
Animals and immunization
Female BALB/c and C57BL/6 mice (between 7 and 9 weeks of age) were purchased from Japan SLC (Hamamatsu, Japan) and maintained at the Institute for Experimental Animals, Hamamatsu University School of Medicine. All animal experiments were performed according to the Guidelines for Animal Experimentation, Hamamatsu University School of Medicine.
Mice were epidermally immunized with plasmids (2 µg per mouse per immunization) using the Helios gene gun system (Bio-Rad Laboratories, Hercules, CA) as described previously (Suzuki et al., 2004) or recombinant proteins (50 µg per mouse) emulsified with incomplete Freund's adjuvant (Difco Laboratories/BD Diagnostic Systems, Sparks, MD) by injecting subcutaneously three times at 10-day intervals.
ELISA for interferon-γ (IFN-γ)
Two weeks after the last immunization, spleen cells from immunized BALB/c and C57BL/6 mice were cultured in a 96-well plate (1 × 106/0.2 cells mL−1) in the presence of MPT51 peptides (1 µM) for 2 and 4 days, respectively. Antigenic peptides P24-32 and P171-190 (a dominant H2-Ab-restricted epitope) (Suzuki et al., 2004) were used for CD8+ and CD4+ T-cell stimulation, respectively. For protein immunization, spleen cells from immunized or naïve C57BL/6 mice were stimulated with an antigenic peptide P171-190 (1 µM) for 4 days as described above. The IFN-γ concentration of the culture supernatants was determined by a sandwich ELISA as described previously (Yoshida et al., 1995).
Preparation of total RNA and semi-quantitative reverse transcriptase (RT)-PCR
Two weeks after the last immunization, spleen cells from immunized C57BL/6 mice were cultured in a 12-well plate (1 × 107 cells mL−1 per well) in the presence of 1 µM MPT51 peptide (P171-190) for 16 h. Total RNA was prepared using ISOGEN (Nippon Gene, Tokyo, Japan), and semi-quantitative RT-PCR was performed as described previously (Uchijima et al., 2005) using primers as follows:
inducible nitric oxide synthase (iNOS)
5′-TACAAGATGACCCTAAGAGT-3′ (forward)
5′-ACATGGCCGAGCGTCAAAGA-3′ (reverse)
5′-TCTGAGACAATAAACGCTAC-3′ (forward)
5′-GAATCAGCAGCGACTCCTTT-3′ (reverse)
glycerol-3-phosphate dehydrogenase (G3PDH)
5′-ACCACAGTCCAT CCATCAC-3′ (forward)
5′-TCCACCACCCTGTTGCTGTA-3′ (reverse)
Statistical analysis
Statistical analyses were performed using statview-j 5.0 statistics program (SAS Institute Inc., Cary, NC). The Mann–Whitney test was used to calculate P-values.
Enhancement of MPT51-specific T-cell responses by full-length HSP70
We first examined the enhancing effect of full-length HSP70 (HSP70F) for the induction of MPT51-specific T-cell responses. Because MPT51 induces only CD4+ or CD8+ T-cell response in C57BL/6 or BALB/c, respectively (Suzuki et al., 2004), we used both strains to investigate their corresponding T-cell responses. C57BL/6 and BALB/c mice were immunized with a fusion gene of HSP70F and MPT51 (HSP70F-MPT51) or MPT51 using the Helios gene gun system. Spleen cells from immunized C57BL/6 and BALB/c mice were used for IFN-γ production assays of CD4+ and CD8+ T cells, respectively, in response to corresponding antigenic peptides. CD4+ T cells from C57BL/6 mice immunized with HSP70F-MPT51 produced a significantly higher amount of IFN-γ than those immunized with MPT51 (Fig. 1). The addition of HSP70F to MPT51 did not show a significant enhancing effect on the antigen-specific CD8+ response, although CD8+ T cells from BALB/c mice immunized with HSP70F-MPT51 showed a slightly higher IFN-γ production than those immunized with MPT51. Immunization with HSP70F DNA alone did not induce MPT51-specific immune responses in C57BL/6 and BALB/c mice, and the amounts of IFN-γ production from spleen cells of immunized mice in response to corresponding peptides were similar to those of naïve mice (data not shown). In contrast to IFN-γ production, HSP70F-MPT51 showed no enhancing effect on MPT51-specific interleukin-4 (IL-4) production in both strains, although spleen cells from both strains similarly produced IL-4 in response to anti-CD3 mAb stimulation (data not shown). Taken together, the addition of HSP70 to MPT51 enhanced antigen-specific T-helper cell type 1 (Th1), but not Th2 responses.
Identification of the domain of HSP70 responsible for its enhancing effect on MPT51-specific T-cell response
Because HSP70 comprises two major domains, that is, the N-terminal ATPase domain (44 kDa) and the C-terminal peptide-binding domain (27 kDa), we next attempted to identify the domain that was responsible for the enhancement of the MPT51-specific T-cell response. For this purpose, we prepared additional fusion genes, HSP70N-MPT51 and HSP70C-MPT51 (Fig. 2a).
Before immunization, 293 T cells were transfected transiently with plasmids that encoded these chimeric MPT51 DNAs, to confirm the protein expression in vitro. Both fusion proteins, as well as HSP70F-MPT51 and MPT51, were detected at around the expected molecular sizes by Western blotting with anti-MPT51 mAb (Fig. 2b), indicating the proper amino acid sequences of expressed proteins.
BALB/c and C57BL/6 mice were then immunized with the plasmids containing HSP70N-MPT51, HSP70C-MPT51, or MPT51 by gene-gun and their spleen cells were tested for MPT51-specific T-cell responses. A stronger IFN-γ production from CD4+ T cells was induced by HSP70C-MPT51 compared with that by HSP70N-MPT51 and MPT51 alone (Fig. 3). However, HSP70C-MPT51 showed no enhancing effect on MPT51-specific IL-4 production (data not shown). Although the CD4+ T-cell response induced by HSP70N-MPT51 seemed stronger than that induced by MPT51, the difference was not statistically significant. In contrast, the addition of either HSP70N or HSP70C did not have any effect on the induction of CD8+ T-cell responses, which confirmed the results with HSP70F. The enhancing effect of HSP70C was confirmed by semi-quantitative RT-PCR (Fig. 4). The expression of mRNA for IFN-γ was increased considerably, which resulted in the upregulation of mRNA for iNOS, probably in APCs. These results together indicate that the C-terminal domain of M. tuberculosis HSP70 contributes to the enhancement of antigen-specific Th1 responses.
Induction of MPT51-specific CD4+ T-cell responses by MPT51-HSP70 fusion proteins
We finally prepared fusion MPT51 proteins and examined their effects on the induction of MPT51-specific CD4+ T-cell responses. Although SDS-PAGE analysis showed all Ni-NTA-purified proteins to have molecular sizes slightly smaller than those algorithmically expected (Fig. 5a), they were confirmed to have proper amino acid sequences by sequencing analyses (data not shown). When mice were immunized with these recombinant proteins, HSP70F-MPT51 and HSP70C-MPT51 again induced stronger immune responses than HSP70N-MPT51 and MPT51, and the effect of HSP70C-MPT51 was superior to that of HSP70F-MPT51 (Fig. 5b). These results confirm the evidence obtained by DNA vaccination that M. tuberculosis HSP70, especially its C-terminal domain, facilitates the induction of antigen-specific CD4+ T-cell responses.
In DNA vaccines, the plasmid DNA is delivered into somatic cells (such as keratinocytes or myocytes) and professional APCs (such as DCs), but the former cells serve as a predominant reservoir for antigen (Gurunathan et al., 2000; Kutzler & Weiner, 2008). DNA vaccines express a low amount of antigen at the restricted site of inoculation, and the frequency of DC bearing vaccinated DNA is very low (Casares et al., 1997; Barouch et al., 2004). Therefore, intercellular antigen spreading seems particularly important to increase the number of APCs. In the present study, we attempted to establish a new fusion DNA vaccine against MPT51 using M. tuberculosis HSP70 as a partner to facilitate the receptor-mediated uptake of shed antigens from transfected somatic cells that underwent apoptosis and/or necrosis by professional APCs. With this new vaccine construct, we demonstrated that HSP70, especially its C-terminal domain, had an enhancing effect on the induction of MPT51-specific CD4+ (but not CD8+) T-cell responses.
Microbial and mammalian HSP70s effectively induce antigen-specific immune responses in various ways (Asea et al., 2000; Kuppner et al., 2001; Somersan et al., 2001), and several receptors have been identified for them, that is, CD91 (Basu et al., 2001), CD40 (Wang et al., 2001; Becker et al., 2002), CCR5 (Floto et al., 2006), TLR-2, and TLR-4 (Asea et al., 2002). Tobian (2004a) have reported that exogenous M. tuberculosis HSP70 can CD91-dependently enhance the presentation capacity for ovalbumin (OVA)-derived major histocompatibility complex (MHC) class I peptides in macrophages and DCs. This has established CD91 as a receptor for prokaryotic as well as mammalian HSPs. On the other hand, they have also reported that the enhancing effect of M. tuberculosis HSP70 on the presentation of MHC class II peptides is CD91-independent (Tobian et al., 2004b). HSP70 has also been reported to bind to a chemokine receptor CCR5 (Whittall et al., 2006) and transduce various signals into DCs to enhance immune responses (Floto et al., 2006). We reported previously that a fusion protein of MIP-1α and MPT51 is internalized preferentially into DCs via CCR5, and consequently, induces stronger MPT51-specific CD8+ and CD4+ T-cell responses than MPT51 alone (Uchijima et al., 2008 and unpublished data). It is possible that M. tuberculosis HSP70 exerts its enhancing effect in the same way, although further analysis is necessary. Alternatively, additional signals via other HSP70 receptors, such as CD40 and/or TLRs, may contribute to the enhancing effects. Taken together, these observations suggest that activation signals transduced via HSP70 receptors other than CD91 play pivotal roles in the enhancement of immune responses, although CD91 may contribute to the internalization of MPT51 fusion proteins conjugated with HSP70.
The domain of HSP70 that is responsible for its enhancing effect is still a controversial subject. Some groups have reported that the C-terminal peptide-binding domain might act as a microbial adjuvant (Wang et al., 2002; Qazi et al., 2005). Wang and colleagues showed that the C-, but not the N-terminal domain upregulates the expression of cytokines and their receptors (IL-12, tumor necrosis factor-α, NO, C–C chemokines, and CCR7), costimulatory molecules (CD83, CD86, and CD80), and HLA class II molecules in human hematopoietic cells. Of these upregulated molecules, IL-12 is known to be a very potent cytokine for Th1 polarization (Trinchieri, 1994), but they showed Th1 skewing of immune responses only by an isotype analysis of immunoglobulins specific for HSP70 itself. Qazi and colleagues also described the effectiveness of the C-terminal domain, but they did not compare its effect with that of the N-terminal domain. Together with these observations, our current findings showing the enhancing effect on targeted-antigen-specific IFN-γ production by CD4+ T cells strengthen the rationale for the use of the C-terminal domain of HSP70 as a Th1-polarizing adjuvant. The enhancing effect of C-terminal domain of HSP70 is more prominent when used in protein immunization. As shown in Fig. 5b, the addition of HSP70C (and HSP70F) to MPT51 effectively induced antigen-specific immune responses, even if the antigen alone could not induce the response. In contrast, Udono (2001) have identified amino acid residues 280–385 in the N-terminal ATPase domain of HSP70 as the most important region for cytotoxic T lymphocyte (CTL) induction. Huang (2000) have also shown that the N-terminal domain of HSP70 (residues 160–370) is sufficient to stimulate the substantial generation of anti-OVA CTL in the absence of an adjuvant. At present, we have no evidence to explain why HSP70N did not show an enhancing effect on CD8+ T-cell responses in our system. Our construct contains the entire structure of the N-terminal domain while other effective constructs lack the extreme N-terminus, and this portion might have an inhibitory effect. Alternatively, the difference(s) in the immunogenicity of antigens (OVA vs. MPT51) and/or strain (C57BL/6 vs. BALB/c) may be accountable for the ineffectiveness. Besides the enhancing effect of the N-terminal domain on immune responses, however, its immunosuppressive role by the production of IL-10 and transforming growth factor-β has also been demonstrated in rats (Kimura et al., 1998; Wendling et al., 2000). In general, the C-terminal peptide-binding domain of HSP70 tends to facilitate CD4+ T-cell responses more effectively than CD8+ T-cell responses, which skews the cytokine milieu toward Th1.
IFN-γ-secreting CD4+ T cells known as Th1 cells are important mediators of tuberculosis protection (Cooper et al., 1993; Flynn et al., 1993), and attempts to induce tuberculosis antigen-specific Th1 cells have been the dominant theme of most tuberculosis-vaccine development (Skeiky & Sadoff, 2006). Although CD8+ CTLs have also been reported to contribute to disease resistance (Flynn & Chan, 2001; Kaufmann, 2003), our current findings may pave the way for the establishment of a novel vaccine against M. tuberculosis.
We thank Ms. Y. Suzuki for her expert assistance. This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, the COE Research and the Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Health and Labour Science Research Grants for Research into Emerging and Reemerging Infectious Diseases from the Ministry of Health, Labour and Welfare of Japan, and the United States–Japan Cooperative Medical Science Committee.