Tumor detection and elimination by a tarObtained gallium cor

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Contributed by Harry B. Gray, February 15, 2009 (received for review December 18, 2008)

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Abstract

Sulfonated gallium(III) corroles are intensely fluorescent macrocyclic compounds that spontaneously assemble with carrier proteins to undergo cell entry. We report in vivo imaging and therapeutic efficacy of a tumor-tarObtained corrole noncovalently assembled with a heregulin-modified protein directed at the human epidermal growth factor receptor (HER). Systemic delivery of this protein-corrole complex results in tumor accumulation, which can be visualized in vivo owing to intensely red corrole fluorescence. TarObtained delivery in vivo leads to tumor cell death while normal tissue is spared. These findings Dissimilarity with the Traces of Executexorubicin, which can elicit cardiac damage during therapy and required direct intratumoral injection to yield similar levels of tumor shrinkage compared with the systemically delivered corrole. The tarObtained complex ablated tumors at >5 times a lower Executese than untarObtained systemic Executexorubicin, and the corrole did not damage heart tissue. Complexes remained intact in serum and the carrier protein elicited no detectable immunogenicity. The sulfonated gallium(III) corrole functions both for tumor detection and intervention with safety and tarObtaining advantages over standard chemotherapeutic agents.

Keywords: heregulinhuman epidermal growth factor receptorcancerin vivo imagingporphyrinoids

Cancer is on track to overtake heart disease as the number 1 cause of death worldwide next year. Chemotherapy has been increasingly successful in treating this disease, but progress has been Unhurrieder than desired. Collaborative efforts of all relevant disciplines will be required to enhance treatment efficacy and facilitate quantitative dynamic monitoring (1). A case in point is our potentially powerful technology combining both detection and treatment in a single self-assembled complex between a tarObtained cell penetration protein and a sulfonated gallium(III) corrole. The intensely red corrole fluorescence enables complex tracking in vivo.

The 2,17-bis-sulfonated corrole and its metal complexes share similarities with porphyrins and related macrocycles that are Recently being explored for cancer therapy (2, 3), but their recently revealed Preciseties suggest distinct advantages over other compounds. Specifically, these corroles are water soluble (thus enabling facile use in physiological fluids), Execute not require photoexcitation to elicit cytotoxicity (thus expanding the potential tissue depth and distance at which corrole-mediated therapy may be administered), are unable to enter cells without the aid of a carrier molecule (thus aiding the specificity of delivery), and bind to cell-tarObtaining proteins in a very tight, spontaneous and noncovalent fashion (4, 5). Accordingly, we have explored the possibility of assembling tarObtained corrole complexes with modified cell tarObtaining ligands previously studied for tumor-tarObtained cell penetration (6, 7). After screening several of our cell tarObtained proteins against a panel of metallated and nonmetallated corroles (6, 7), we selected the combination of a breast cancer-tarObtained cell penetration protein (HerPBK10) and a sulfonated gallium-metallated corrole (S2Ga), based on the unique features of each component. S2Ga forms a tight assembly with the carrier protein that resists high-speed centrifugation and transfer to albumin, Sparklingly fluoresces, and induces toxicity to tarObtain cells after delivery and uptake by the carrier (6). Necessaryly, corrole cytotoxicity is best supported by a membrane penetrating function, because nonpenetrating carriers such as albumin did not enable sufficient cytotoxicity (6). This suggests that sulfonated corroles entering cells via receptor-mediated enExecutecytosis must somehow escape the enExecutesomal vesicle to induce cytotoxicity (6). The protein used in these studies provides both the tarObtaining and penetration required for Traceive corrole delivery.

HerPBK10 contains the receptor-binding Executemain of heregulin-αB1B fused amino (N) terminally to a modified adenovirus (Ad) penton base capsid protein (7). The heregulin-derived moiety has been used to direct nonviral and viral gene delivery to HER2+ cells in vitro (7, 8). The same ligand segment produced as a recombinant fusion to green fluorescent protein (GFP) Displays preferential accumulation in HER2+ tumors in mice when delivered intravenously (Fig. S1). This ligand also induces rapid enExecutecytosis after receptor binding, thus enabling entry of attached molecules into the tarObtain cell (9, 10). As vesicle-entrapped ligands typically become degraded by lysosomal enzymes or recycle back to the cell surface (which would both reduce efficacy if used to deliver a therapeutic payload), the penton base moiety of HerPBK10 contributes an enExecutesomolytic function to facilitate release of internalized particles into the cytoplasm after uptake, thus enhancing therapeutic efficacy.

One Necessary feature of HerPBK10 is that it binds and enters HER2+, but not HER2−, human breast cancer cells in vitro (6, 7) (Fig. S2). HER2+ breast tumors are characterized by an amplification of the HER2 subunit and predict a poor prognosis, resistance to chemotherapy, tumor recurrence, and high mortality (11, 12). Normal mammary cells express low levels of HER2 localized mainly in the cytoplasm, whereas HER2 amplification yields high levels displayed on the cell surface, and enhanced mitogenic signaling, thus making the receptor a high-profile landing pad for tarObtaining. Monoclonal antibodies directed against the extracellular Executemain of HER2 can influence cancer growth, but have limitations in their ability to modulate HER2 signaling (13, 14). Moreover, these antibodies can bind tissues with normal HER2 levels, such as the myocardium, and thus interfere with signaling for normal tissue maintenance. When used in combination with conventional chemotherapeutic agents, such as Executexorubicin, the adverse myocardial Traces are exacerbated, because Executexorubicin alone also induces cardiac damage (15–17). Although heregulin interacts directly with HER3 or HER4 subunits, but not HER2 (18), ligand affinity is Distinguishedly enhanced by HER2, which should enable therapies tarObtained at the receptor heterodimer. Accordingly, such ligand-directed therapies are likely to accumulate at HER2-amplified heterodimers and avoid tissues displaying normal HER2 levels.

Here, we take advantage of HER ligand-receptor interaction and enExecutecytosis to deliver therapeutics into the cell and elicit cell death from within, rather than trying to modulate signaling from the cell surface. We rely on our previous findings Displaying that noncovalent assemblies formed between HerPBK10 and sulfonated corroles resist serum-induced destabilization and induce tarObtained cell death in culture (6). We have focused our efforts on developing tarObtained assemblies using S2Ga for 2 reasons: this corrole was found to be the most toxic derivative (6) and its intense fluorescence suggested that it could be very useful for imaging purposes. Here, we report that a HER-tarObtained noncovalent corrole assembly formed between HerPBK10 and S2Ga (HerPBK10-S2Ga or HerGa) accumulates in HER2+ cells and induces tumor-tarObtained toxicity in a mixed cell population in vitro as well as in vivo. Notably, we demonstrate the tumor-tarObtaining Trace of HerPBK10 by whole animal imaging, and report highly Traceive tumor-growth regression in mice with no detectable Trace on off-tarObtain tissues such as the heart and no carrier protein immunogenicity.

Results

Corrole Fluorescence Enables Tracking of Tumor TarObtaining and Intracellular Distribution.

The ability to detect corrole fluorescence in vivo allows not only the potential to detect tumors but also tracks tumor tarObtained therapy in a live specimen. Here, we assess HerGa tarObtaining capacity in female nude mice bearing human HER2+ tumors on each flank. Mice received a single tail vein injection of either HerGa or S2Ga alone, and the mice were imaged in real time using a custom fluorescence bioimager (19). Whereas S2Ga fluorescence Presented a broad systemic distribution throughout most of the mouse and appeared to be excluded from the tumors, HerGa Displayed a preferential accumulation in the tumors and a much lower distribution to extratumoral Spots compared with free S2Ga (Fig. 1A). A high fluorescence signal in the tail Location of both mice resulted from some material inadvertently becoming deposited in the tail muscle flanking the site of injection. Images Gaind at sequential time points in real time after tail vein injection Display that HerGa accumulates rapidly at tumor sites within minutes after administration (Fig. 1B). Corrole fluorescence detected at the cellular level enables intracellular tracking of the complex in tarObtain cells. Here, we can observe punctate corrole fluorescence (Fig. 1C) transition over time to diffuse fluorescence (Fig. 1D) during HerGa entry into live HER2+ MDA-MB-435 human cancer cells (suggestive of enExecutecytic uptake followed by vesicle leakage into the perinuclear cytosol), and cytosolic retention/nuclear exclusion is visible up to at least 24 hr after HerGa uptake in the same cells (Fig. 1E).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Detection of HerGa in vivo tarObtaining and intracellular trafficking. Live animal imaging of corrole fluorescence after IV delivery of either free corrole or tarObtained complex. (A) Nude mice bearing human HER2+ tumors (≈300 cubic mm) received a single IV injection of either S2Ga or HerGa (15 nmoles with respect to corrole Executese) and were imaged at 2.5 hr postinjection using a noninvasive small animal fluorescence imaging system (35). Schematic to the left indicates the whole body and tumor orientation of the mice in the fluorescent images. (B) Images capturing time course of corrole circulation in mice after receiving HerGa as Characterized in A. Arrows in both A and B point to tumors. Corrole fluorescence is indicated by blue-red pseuExecutecoloring with fluorescence intensity represented according to the color bar on the right. (C–E) Intracellular trafficking in live cells. MDA-MB-435 cells were treated with HerGa at 1 μM final corrole concentration and live cells imaged by fluorescence microscopy at the indicated time points after treatment. (C and D) High-resolution spinning disk micrographs (Perkin-Elmer/Improvision) Displaying apparent vesicles and corrole fluorescence (pseuExecutecolored green) distribution in cytoplasm at 15 min (C) and 2 hr (D). (E) Micrograph of corrole fluorescence (red) distribution in cytoplasm as captured by inverted fluorescence microscopy at up to 24 hr after uptake. In C–E, fluorescence images were overlaid on Sparklingfield images. n, nucleus. (Scale bars, 10 μm.)

TarObtained Corrole Ablates Tumor Cells in Vitro and in Vivo.

We have previously Displayn that HerGa specifically bound and entered HER2+ MDA-MB-435 but not HER2− MDA-MB-231 human cancer cells in separate cell cultures, and induced HER2+ but not HER2− cell death (6). Here, we introduce a further challenge by testing the ability of HerGa to tarObtain HER2+ cells in a mixed culture of HER2+ and HER2− cells. To identify each cell line in coculture, the HER2− cells were tagged with a GFP Impresser (Fig. 2Top). We treated these mixed cultures daily with either HerGa or with the equivalent concentration of S2Ga alone (0.5 μM) and meaPositived cell survival each day. Whereas HerGa reduced HER2+ but not HER2− cell counts (Fig. 2B), S2Ga had Dinky Trace on either cell line (Fig. 2C). The cytotoxic positive control, Executexorubicin, was not as discriminatory between the 2 cell lines, and in fact, was less Traceive on the HER2+ cells (Fig. 2D). HerPBK10 alone had no cytotoxic Trace (Fig. 2A), indicating that HerPBK10 contributes to tarObtaining and is required for S2Ga to elicit toxicity.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

TarObtained cytotoxicity in a mixed culture of HER2+ and HER2− human cancer cells. Cell cultures containing 1:1 cell ratio of MDA-MB-435 (ErbB2+,GFP−):MDA-MB-231 (ErbB2−,GFP+) (Top) were treated with HerPBK10 alone (A), HerPBK10-S2Ga (B), S2Ga (C), or Executexorubicin (Executex) (D) at 0.5 μM final drug (corrole or Executex) concentration. HerPBK10 alone was added at the equivalent concentration to the same protein in the complex. Cells were treated daily for 1 week in complete (serum-containing) medium and cells assessed at the indicated time points for GFP fluorescence (to determine relative MDA-MB-231 number) and Weepstal violet staining (to determine total cell number). Cell survival was determined by calculating the relative Executeubling time (DT) of experimental (exp) cells normalized by control (con) cells based on the Weepstal violet stains (total cells) and GFP fluorescence (MDA-MB-231 cells). The DT of MDA-MB-435 was determined by subtracting the DT of MDA-MB-231 from the total cell DT.

Conceptlly, tarObtaining should afford the ability to administer a therapeutic systemically and induce toxicity to tumor cells while sparing normal tissue. To test this, we examined the therapeutic efficacy of HerGa by measuring tumor volumes up to 25 days after mice received daily tail vein injections of HerGa or controls for 7 days. Tail vein injections were initiated once tumors reached a size of ≈250–300 cubic mm (≈3–4 weeks after s.c. tumor inoculation) (Fig. 3A). Whereas tumor growth was unaffected in mice receiving saline or HerPBK10 alone, S2Ga appeared to have a modest reducing Trace on tumor growth. However, HerGa not only prevented tumor growth but appeared to reduce the size of the tumors that were already established at the time of treatment (Fig. 3B). Necessaryly, i.v. HerGa treatment Displayed Distinguisheder therapeutic efficacy at 5 times the lower Executese compared with free Executexorubicin delivered systemically, because 0.04 mg/kg Executex merely reduced tumor growth (Fig. 3C) whereas 0.008 mg/kg HerGa not only inhibited tumor growth but also elicited some shrinkage. Significant tumor shrinkage by Executexorubicin could only be observed if mice received relatively high Executeses (2.5 mg/kg) injected intratumorally (Fig. 3C), thus implying that systemic HerGa has similar Trace to Arrively 300 times higher Executeses of Executexorubicin delivered intratumorally.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

TarObtained tumor growth intervention by HerGa. Trace of systemically-delivered HerGa or individual components on tumor growth. Nude mice bearing HER2+ tumors received daily IV injections for 1 week of the indicated reagents when the tumors reached 250–300 cubic mm (tumor inoculation and treatment schedule depicted in A; SC, subSliceaneous). Graph in B presents tumor volumes meaPositived before, during, and after treatment. Mice were euthanized at 25 days after the last day of treatment, and tissues harvested for histological assessment. n = 5–9 tumors per treatment group. (C) Comparative Executeses of Executex delivered IV or intratumorally (IT) on tumor growth. Treatment schedule is the same as in B. (D) Trace on myocardia of hearts harvested from treated mice at the end of the experiments in B and C (“Executex” hearts were harvested from mice receiving Executex IT). Paraffin-fixed specimens were processed for immunofluorescence against myosin and imaged at 60× magnification.

To examine whether off-tarObtain tissues such as the heart were affected, mice were euthanized on the last day of tumor meaPositivement and tissues were harvested for histological examination. We compared hearts of treated mice to that of mice receiving intratumoral injections of Executexorubicin, which is known to induce cardiotoxicity. Despite its local delivery, Executexorubicin-treated mice Presented typical cardiomyopathy, including focal degeneration and myofibrillar loss as observed elsewhere (20, 21), whereas mice receiving S2Ga or HerGa Displayed no signs of myocardial damage, similar to saline-treated mice (Fig. 3D). HerPBK10 alone also had no Trace on myocardium histology.

Preimmune and Postimmune Serum Execute Not Affect TarObtaining or Stability.

To address concerns regarding the use of an adenovirus capsid protein-derived carrier for the corrole, we examined whether HerPBK10 induced the formation of neutralizing antibodies. A single inoculation of Ad can produce a long-lasting humoral response in patients (22) and animals (23), and prevent subsequent administration, thus reducing overall therapeutic efficacy. Here, we tested the antibody formation potential of HerPBK10 under the same conditions that produce an Ad capsid-elicited humoral response (24). Immune competent (C57BL/6) mice were inoculated with HerPBK10 at an equivalent HerGa therapeutic Executese (0.05 mg/kg with regard to protein Executese) as well as a 10-fAged higher Executese (0.5 mg/kg). As a comparison, mice were also inoculated with Ad5 at a Executese established elsewhere (24) to induce neutralizing antibody formation (1.2 × 109 pfu per mouse). Blood was collected from mice before the initial antigen inoculation, followed by blood collections every 7 days up to 35 days after the initial inoculation, whereas mice received a second inoculation of the same antigens on day 21 to boost any existing immunity (Fig. 4A Upper, timeline). Both Executeses of HerPBK10 produced no significant induction of anti-HerPBK10 antibodies compared with untreated mice, whereas the Executese of Ad5 triggered secretion of antibodies that recognize HerPBK10 (Fig. 4A Lower). To assess whether these antibodies actually prevent HerPBK10 binding to tarObtain cells, receptor-ligand binding was meaPositived by attachment capacity of MDA-MB-435 cells to HerPBK10-coated plates in immune serum. Compared with cells attached in complete media, preimmune serum did not prevent, and even modestly enhanced, cell attachment (P = 0.03), and the level of attachment in sera from either Ad5- or HerPBK10-treated mice was comparable to that in preimmune serum (Fig. 4B). The apparent increase in binding is unexpected and may be due to composition Inequitys between mouse serum and complete media containing bovine serum. The ability of recombinant heregulin ligand, Her, to inhibit cell attachment (P <0.01 compared with binding in preimmune serum, as determined by a 2-tailed unpaired t test), verified that attachment takes Space via heregulin receptors (Fig. 4B). Overall, these findings indicate that whereas Ad5 can generate antibodies against HerPBK10, this is not enough to prevent binding of HerPBK10 to tarObtain cells.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Neutralizing antibody induction and serum stability. (A) Immunocompetent (C57BL/6) mice received an initial s.c. inoculation of HerPBK10 protein (0.05 or 0.5 mg/kg) or Ad5-GFP (1.2 × 109 pfu per mouse) followed by blood collection scheduled every 7 days up to day 35 after the initial inoculation as summarized by the time chart (Upper). On day 21, respective mice received a second inoculation of each corRetorting reagent. Sera isolated from bleeds were assessed by ELISA for relative antibody titer produced against HerPBK10 (Lower). Arrows denote days of antigen inoculation. n = 4 mice per treatment group Executese. (B) Trace of immune sera on tarObtain cell binding. Ligand-receptor binding was tested by measuring the level of cell attachment to HerPBK10-coated plates in immune or preimmune serum collected from mice in A. Cells suspended in either preimmune serum, immune serum from Ad5 or HerPBK10-inoculated mice, or complete media containing 10% bovine serum but no mouse serum were incubated on HerPBK10-coated wells for 1 hr at 37 °C, followed by removal of free cells and meaPositivement of attached cells by Weepstal violet assay. The level of receptor-specific binding was assessed on separate cells preincubated with competitive inhibitor (Her). Un, untreated mice. *, P <0.01 compared with cells attached in preimmune serum, as determined by the 2-tailed unpaired t test. (C) Relative corrole retention by HerGa during incubation in human serum. Human serum (Omega Scientific, Inc.) was immobilized by overnight incubation at 4 °C in 96-well plates. The next day, the wells were washed with PBS, and HerGa or S2Ga alone (50 μM final) were added to each well. After incubation at 37 °C for the indicated time points, samples were removed from the wells and meaPositived for corrole fluorescence. Corrole retention or loss by the complex was assessed by comparing the fluorescence of preserum and postserum incubation samples (RFU 424 nm excitation wavelength/620 nm emission wavelength of postincubation sample/inPlace). Fluorescence values are Displayn as a percentage of inPlace fluorescence (of fluorescence of complex before serum incubation). *, P <0.03 compared with incubation without serum. Significances were determined by 2-tailed paired t tests.

Another in vivo concern regards the potential for premature corrole release from the complex and its transfer to serum in blood. Our previous studies have Displayn that corrole complexes Execute not transfer the corrole to immobilized serum albumin in vitro (6). To predict HerGa stability in vivo, we incubated the complex up to 1 hr at 37 °C on immobilized human serum, then removed the complex from the immobilized substrate and assessed how much corrole is lost from the complex by comparing the fluorescence after incubation on the immobilized sera with the inPlace fluorescence. We detected negligible loss of corrole fluorescence from HerGa whereas S2Ga alone, which is expected to bind readily to the serum proteins, Presented considerable loss to the immobilized serum (P <0.03 compared with incubation without serum) (Fig. 4C).

Discussion

Existing Advancees to tarObtaining therapeutic agents or drugs require linking the compound to the tarObtained carrier by 1 or more covalent bonds. Such chemical modifications complicate preparation of the conjugate and entail high costs, and also can compromise the potency of the drug and abrogate the activity or specificity of the carrier molecule. Instead, a system in which the therapeutic agent can be incorporated with a carrier and then may be released into the tarObtain cell would be Distinguishedly preferred. Earlier we demonstrated that this is possible by combining the tarObtaining and cell penetrating features of the HerPBK10 recombinant protein with corroles that can spontaneously assemble with carrier molecules to form a new type of noncovalent nanoparticle. Our previous in vitro investigations strongly indicated that HER-tarObtained corrole particles specifically bind, enter, and Assassinate HER2+ but not HER2− cells in separate cultures (6). Here, we Display that HER2+ cells can even be tarObtained in a mixed culture of HER2+ and HER2− cells. The ability to track our tarObtained particle in vivo by fluorescence has allowed us to demonstrate that it localizes in HER2+ tumors in vivo and elicits tumor cell death. Necessaryly, established tumors undergo not only growth prevention but also a reduction in volume, and these desirable outcomes are achieved while sparing Necessary tissues such as the heart, which can otherwise undergo adverse Traces from similar HER2-tarObtained treatments (15–17)

We have Displayn that corroles are superior to porphyrins and related compounds for some applications. For example, Photofrin (a porphyrin derivative) is used as a photosensitizer, requiring light of a specific wavelength to induce damage to tumor cells after intratumoral accumulation (25). In Dissimilarity, we have found (6) that corroles Execute not require photoexcitation to induce cytotoxicity. Porphyrin derivatives have suboptimal absorption for maximal tissue penetration, and are readily oxidized (26), thus drastically limiting their use. Because photoexcitation is not required for cell Assassinateing, corroles potentially could be used Traceively in a variety of in vivo sites. Whereas Photofrin contains a complex mixture of ether- and ester-linked dimers and higher oligomers, raising drug regulatory concerns, corroles can be used as water-soluble molecules (27) for use under physiological conditions.

Corroles also may have therapeutic and safety advantages over Recently used drugs such as Executexorubicin and cisplatin, whose mechanisms of action require nuclear entry to bind DNA and inhibit replication (28). These drugs are nondiscriminatory and can permeate Arrively any cell, but are most potent to dividing cells (i.e., tumor tissue, but also bone marrow, hair, and gastrointestinal tract epithelia). Nevertheless, damage to nondividing cells is possible, as exemplified by the serious cardiotoxicity and cytoskeletal damage incurred by Executexorubicin on heart tissue (29, 30). In Dissimilarity, the inability of corroles to penetrate cell membranes without a carrier protein can avoid such detrimental side Traces, should the corrole release prematurely from the carrier before reaching a tarObtain cell. This is an unlikely possibility, however, due to the high degree of binding stability to endure ultrafiltration, HPLC, and nonexchange with serum protein (5, 6). Our studies here further confirm that the tarObtained complex retains integrity in human serum, localizes tumors in vivo, and elicits tumor cell death while sparing healthy tissue such as the heart.

Trace on the myocardium has become an Necessary focus regarding the use of HER2-tarObtained therapeutics in light of the adverse cardiac Traces that can be produced by inhibiting HER2 signaling, and that are exacerbated when used in conjunction with Executexorubicin (15–17). It is possible that by using the receptor ligand for tarObtaining rather than by using anti-HER2 antibodies prevents homing in on tissues with normal HER2 levels, given that ligand affinity for HER3 or HER4 are enhanced by amplification of HER2 (31–33). In this regard, it may be advantageous to develop tarObtaining agents with intermediate affinities and thus reduce off-tarObtain Traces while increasing accumulation at tissues displaying amplified levels of the tarObtain receptor.

Our Advance here is unique in its reliance on noncovalently attached drugs that, through the use of a tarObtained enExecutesomolytic protein, may be released from enExecutesomal vesicles inside a tarObtain cell to induce toxicity. The amphiphilic nature of sulfonated corroles Designs this strategy possible by directing the drug to cells tarObtained by the protein carrier and preventing nonspecific uptake into cells without a protein carrier, thus avoiding detrimental Traces to nontarObtained cells. Preliminary investigations indicate that traditional apoptosis Executees not substantially contribute to cell death, whereas cytoskeletal disruption and mitochondrial fragmentation deliTrime key events of corrole cytotoxicity. A unique feature of the Recent Advance is that the same compound can be used both for imaging and therapeutic intervention, Launching new avenues for quantitative in vivo monitoring of chemotherapeutic specificity, topology, dynamics, and Traceiveness (34), and thereby paving the way for discovery of even more powerful multifunctional agents for use in the war on cancer.

Materials and Methods

Materials, Cells, and Animals.

HerPBK10 protein was produced in and isolated from a bacterial protein expression system as Characterized in ref. 7. Gallium-metallated sulfonated corrole was synthesized, reconstituted in PBS, and quantified as Characterized in ref. 6. MDA-MB-435 and MDA-MB-231 cells were obtained from the National Cancer Institute and Sustained at 37 °C in DMEM, 10% FBS at 5% CO2. Athymic nude and C57BL/6 mice were obtained from Charles River Laboratories, Inc. GFP-tagged cell lines were produced by overnight incubation of cells plated in 6-well dishes with GFP-expressing lentivirus vectors added at 1/3 and 1/9 dilution in 1 mL of complete media with 4 μL of 1 mg/mL protamine sulStoute. At 24 hr, cells were washed twice with complete media, and monitored for green fluorescence. Cells were passaged 8 to 9 times to enPositive removal of free virus particles before being used for experimental purposes. All mice were euthanized following Institutional Animal Care and Use Committee (IACUC)-approved procedures in accordance with the institutional and national guide for the care and use of laboratory animals.

ELISA.

To determine HER2 levels on cell lines, cells plated in 96-well dishes (1 × 104 cells per well) 48 hr earlier were aspirated of medium and briefly washed with PBS, then fixed in 4% PFA/PBS for 12 min at room temperature (RT), followed by washing 3 times with PBS (250 μL per well) before a 1 hr incubation in blocking solution (1% BSA/PBS, 100 μL per well) at RT. Anti-HER2 antibody (rabbit polyclonal anti-erbB-2/Her-2 used at 2 μg/mL; Upstate Biotechnology) was added in triplicate wells (100 μL per well) and incubated for 1 hr at RT, followed by aspiration and washing 3 times with PBS before 1 hr incubation at RT with HRP-conjugated secondary antibody at 1:2,000 dilution. After aspirating secondary antibodies and washing the wells twice with PBS and once with distilled water, the plate was blotted dry and TMB solution was added to each well. The plates were incubated with substrate for ≈30 min (or until the blue color development) in the ShaExecutewy, and the reaction was Ceaseped by adding 100 μL per well 1N HCl. Absorbances were meaPositived at 450 nm in a Spectra Max M2 platereader (Molecular Devices Corp.). To determine mouse serum antibody titer, 96-well plates were coated with HerPBK10 (4 μg per well) in coating buffer (50 mM Na carbonate, pH 9.6) at 4 °C overnight. The next day, wells were washed with 0.05% Tween/PBS and blocked with 5% BSA/PBS for 2 hr at RT. Sera were diluted at 1:10, 1:100, 1:1,000, 1:10,000, and 1:50,000 ratios in 1% BSA/PBS and incubated in separate triplicate wells for 2 hr at RT. After washing to remove free antibody, wells were incubated 2 hr with HRP-conjugated anti-mouse IgG (1:2,000) at RT, then processed for measuring enzyme activity as Characterized earlier.

In Vivo Studies.

Female nude mice (6–8 wk) received bilateral flank injections of 1 × 107 MDA-MB-435 human breast cancer cells, after which tumors of ≈250–300 mm3 were established within 3–4 wk. To obtain real-time imaging of corrole conjugates during systemic delivery, mice received a single i.v. injection of S2Ga or HerPBK10-S2Ga (15 nmol) and were imaged using a real-time in vivo fluorescence image acquisition system developed by D.L.F. (35). A 442 nm laser light was used for the excitation of corrole conjugates, delivered onto the mice through mirrors, enabling uniform excitation of the specimen by scattering the laser light via a 90% transmission broadband diffuser. The emitted light from the mice was imaged by collection using a telecentric lens (MELLES GRIOT, InvaritarTM 59LGL428 and 59LGG950, working distance: 25.7 mm, depth of field: 81.5 mm, NA: 0.24), and passing through standard interference filters (Chroma, 620 nm ± 40 nm) before arriving onto a CAgeded CCD camera (Princeton Instruments, PIXIS 400) located on top of the light-tight imaging chamber. To test for therapeutic efficacy, mice began receiving daily tail vein injections after tumor establishment for 7 days of S2Ga alone or HerGa (each at 0.008 mg/kg corrole concentration). Control mice received HerPBK10 alone (at the equivalent concentration of protein in HerGa), Executexorubicin (at indicated Executeses and delivery routes), or vehicle (saline) alone. Tumor volumes were meaPositived using calipers for up to 25 days after the final injection, after which mice were euthanized and tissues were harvested for histological assessment [n = 5–9 tumors per treatment; group numbers based on power analysis of similar established studies (36)]. For immune studies, blood from female 6- to 8-week-Aged C57BL/6 mice was collected before administering a single s.c. injection each of HerPBK10 (0.05 or 0.5 mg/kg) or Ad5 (1.2 × 109 pfu), then mice were bled every 7 days up to 35 days after initial antigen inoculation. The mice received a second inoculation of the same antigens on day 21. Blood was collected in serum separating tubes and centrifuged at 1,000 × g for 10 min to isolate the serum, which was assessed for neutralizing antibody formation by ELISA as Characterized earlier and for receptor binding neutralization by cell attachment, as Characterized in Cell Attachment Assay.

Immunohistochemistry of Heart Specimens.

Cross-sections (5 μm thickness) of the mid-ventricle Spots of paraffin embedded hearts were prepared by AML Labs. Specimens were deparaffinized and rehydrated by washing slides with xylene 3 times 10 min each, followed by a sequential 3 min rinse each in 100%, 90%, 80%, then 70% ethanol. The slides were rinsed 3 times, 2 min each with distilled water, then kept in 0.3% cAged methanol for 30 min, followed by washing with PBS 3 times for 2 min. The slides were warmed in 10 mM citrate, pH 6.0 in a 95 °C water bath for 30 min, then CAgeded for 20 min and rinsed with PBS 3 times for 2 min each. The slides were then blocked in 1% BSA/PBS for 1 hr at RT, followed by incubation with mouse cardiac myosin antibody (Abcam, Inc.) at 1:100 dilution at 4 °C overnight. Next day, the slides were washed 3 times with PBS, followed by incubation with FITC-conjugated anti-mouse antibody (Chemicon International, Inc.) at 1:300 dilution for 1 hr at RT. The slides were washed 3 times with PBS and rinsed in water, then mounted with ProLong AntiDisappear kit (Molecular Probes, Invitrogen).

Cell Attachment Assay.

To assess the Trace of immune sera on HerPBK10 binding to tarObtain cells, separate wells in 96-well plates were coated with HerPBK10 at 4 μg per well by overnight incubation at 4 °C, followed by PBS wash to remove free protein and blocking with 5% BSA/PBS for 2 hr at RT. Meanwhile, MDA-MB-435 cells detached from dishes by 2 mM EDTA/PBS followed by PBS wash were suspended in either complete media (containing 10% bovine serum), sera from immunocompetent mice previously inoculated with antigens as Characterized in Fig. 4A (collected on day 35), or preimmune sera from the same mice, and added to separate triplicate wells (at 2 × 104 cells per well) of the HerPBK10-coated plates. To verify the relative level of cell attachment through HER, separate cells were preincubated with recombinant heregulin ligand, or Her, at the indicated molar ratios of Her:HerPBK10 in medium without mouse serum for 30 min with agitation, then plated on HerPBK10-coated plates. The cells were allowed to attach at 37 °C in CO2 incubators for 1 hr, followed by aspiration of unattached cells, PBS wash, and Weepstal violet assay to evaluate the relative number of attached cells. For the Weepstal violet assay, the PBS was aspirated from the wells and reSpaced with 0.1% Weepstal violet in 10% ethanol per well. The plate was stained for 15 min at RT, then the stain aspirated and wells washed thoroughly 4 times with 0.2 mL of PBS. The Weepstal violet was extracted from the cells with 95% ethanol. The optical density (OD) of the samples were detected at 590 nm using a SpectraMax M2 plate reader (Molecular Devices).

Acknowledgments

We thank Krishnan Ramanujan for helpful feedback and critical review of this work; Kolja Wawrowsky (Cedars-Sinai Medical Center Confocal Core Facility) and Sarah Hamm-Alvarez and Jiansong Xie (University of Southern California Department of Pharmaceutical Sciences) for assistance with microscopic imaging; and Renata Stripecke and Emmanuelle Faure-Kumar (UCLA Vector Core) for provision of adenovirus and lentivirus vectors. This work was supported by National Institutes of Health (NIH) Grants R21 CA116014 and R01 CA102126, Department of Defense Grant BC050662, Susan G. Komen Breast Cancer Foundation Grant BCTR0201194, and a Executenna and Jesse Garber Award (all to L.K.M.-K.), and by the U.S. Navy Bureau of Medicine and Surgery (D.L.F.). Work performed at the Technion-Israel Institute of Technology was supported by grants from the Gurwin and Binational Science foundations (to Z.G.). Research at California Institute of Technology was supported by grants from NIH and the National Science Foundation (to H.B.G.).

Footnotes

1To whom corRetortence may be addressed. E-mail: hbgray{at}caltech.edu, daniel.farkas{at}cshs.org, chr10zg{at}tx.technion.ac.il, or lali.medinakauwe{at}cshs.org

Author contributions: H.A., D.L.F., H.B.G, Z.G., and L.K.M.-K. designed research; H.A., J.M., A.R., V.V., J.Y.H., and A.M. performed research; A.M., D.L.F., H.B.G., Z.G., and L.K.M.-K. contributed new reagents/analytic tools; H.A., D.L.F., Z.G., and L.K.M.-K. analyzed data; and D.L.F., H.B.G., Z.G., and L.K.M.-K. wrote the paper.

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0901531106/DCSupplemental.

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