In vivo anticancer activity of rhomboidal Pt(II) metallacycles

Significance

The design of new antitumor agents and the search for delivery vehicles for existing drugs are two problems in cancer therapy that are often solved separately. Drug delivery vehicles are typically nontherapeutic, leaving antitumor effects exclusively to the encapsulated drug. However, the combination of an anticancer drug and a delivery vehicle with inherent antitumor properties could produce a synergistic effect, thus enhancing the therapy. We describe herein the synthesis and characterization of two supramolecular coordination complexes with well-defined geometries, along with their in vitro localization and in vivo assessment of antitumor effects. These complexes are stable in cell culture and nontoxic to cells, yet show substantial reduction of the tumor growth and tumor burden in mouse xenograft models.

Abstract

The development of novel antitumor agents that have high efficacy in suppressing tumor growth, have low toxicity to nontumor tissues, and exhibit rapid localization in the targeted tumor sites is an ongoing avenue of research at the interface of chemistry, cancer biology, and pharmacology. Supramolecular metal-based coordination complexes (SCCs) have well-defined shapes and geometries, and upon their internalization, SCCs could affect multiple oncogenic signaling pathways in cells and tissues. We investigated the uptake, intracellular localization, and antitumor activity of two rhomboidal Pt(II)-based SCCs. Laser-scanning confocal microscopy in A549 and HeLa cells was used to determine the uptake and localization of the assemblies within cells and their effect on tumor growth was investigated in mouse s.c. tumor xenograft models. The SCCs are soluble in cell culture media within the entire range of studied concentrations (1 nM–5 µM), are nontoxic, and showed efficacy in reducing the rate of tumor growth in s.c. mouse tumor xenografts. These properties reveal the potential of Pt(II)-based SCCs for future biomedical applications as therapeutic agents.

Molecular assemblies of nanoscale-size and well-defined geometries have recently emerged as an interesting new paradigm in drug design and drug delivery. To date, liposomes, the self-assembled lipid nanoparticles held together by weak interactions, are among the most widely studied and clinically successful nanoparticle-based drug carriers. Their use allows the drug to achieve sustained plasma levels while encapsulated, with the size preventing the fast clearance by the kidneys that often occurs with the free drug. However, liposomes themselves do not produce a therapeutic effect and their application as drug carriers for medical purposes has often been hindered by poor loading capacity (<5 wt %) and the inability to pass through biological barriers (1, 2). Inorganic and hybrid porous materials, such as molecular organic frameworks (MOFs), have also shown promise due to their higher loading capacities (>25 wt %) (3–5), but MOFs have poor hydrolytic stability (6, 7). Recent studies on materials from Institut Lavoisier (MIL)-100(Cr) and MIL-100(Fe), however, suggest that MOFs can persist in biologically relevant environments and can act as vehicles for some anticancer and antiviral agents (8–10). These early findings have prompted further investigations into the biomedical applications of supramolecular coordination complexes (SCCs) (11–24). SCCs preserve the attractive properties of MOFs, such as building block modularity (22, 23, 25), yet afford an increased solubility in the biological milieu and lend themselves to small-molecule characterization techniques due to their well-defined structure.

Although development of SCCs for biomedical applications is in its infancy, some SCCs, such as trigonal prisms self-assembled from p-cymene and ruthenium-based metal fragments with pyridyl donors, have shown the ability to act as effective carriers of some chemotherapeutic agents (26–28). Moreover, a library of cytotoxic to cancer cells p-cymene ruthenium-based polygons and cages has also been developed (11). For biomedical applications, the information about the cellular uptake, delivery of a guest, and metabolism of the drug delivery vehicle is critical, although currently the fate of SCCs in biological environments is not well understood. In a rare report, a systematic investigation of the structural stability of a water-soluble, hexacationic ruthenium-based trigonal prism was performed; however, it was determined that the ruthenium-based trigonal prisms decompose in the presence of amino acids histidine, lysine, and arginine (29).

An intriguing approach is the design of tumor-targeted modalities that combine detection and treatment through the self-assembly of emissive, metal-based coordination complexes. Such modalities can be especially valuable as they often do not require photoexcitation to elicit cytotoxicity. Recently Gray, Gross, and Medina-Kauwe and coworkers reported HerGa, a self-assembled tumor-targeted particle that bears the Ga(III)-metalated derivative of the sulfonated corrole (30, 31). The particle, which contained Ga(III)-corrole noncovalently bound to the tumor-targeting cell penetration protein HerPBK10, provided both tumor detection and elimination. Systemic injection of this protein–corrole complex resulted in tumor accumulation, which can be visualized in vivo due to the red corrole fluorescence. Cytotoxic and cytostatic properties of these targeted Ga(III) corroles were found to be cell-line dependent, with the ability to induce late M-phase arrest in several cancer cell lines (32).

Despite the well-known cytotoxic properties of mono- and multinuclear platinum complexes (33–35), studies of the antitumor properties of platinum-based SCCs are rare (17, 36). Moreover, recent reports have demonstrated that platinum-based SCCs can act as effective hosts for guests and have interesting photophysical properties (37–42). In particular, highly emissive rhomboids based on aniline-containing donors and Pt-based metal acceptors have been developed that display different photophysical properties from those of their constituent subunits (40). These assemblies are interesting targets to investigate the cytotoxicity of organoplatinum SCCs, whereas their emission spectra could be used for interrogating the structural integrity in vitro. Here, for the first time to our knowledge, we report the uptake of SCCs in vitro in cell-based assays, determined by using laser-scanning confocal microscopy, and an in vivo assessment of the anticancer activity of SCCs in mouse s.c. tumor xenograft models.

Results and Discussion

Preparation of Rhomboidal Pt(II) Metallacycles.

The synthesis of SCCs is depicted in Fig. 1. Mixing of 2,6-bis(pyrid-4-ylethynyl) aniline 1 and 2,9-bis[trans-Pt(PEt₃)₂NO₃] phenanthrene 3 in methanol at 50 °C for 24 h produced endohedral amine-functionalized Pt-based SCC rhomboid 4 (39). A similar reaction of 1,6-bis(pyrid-4-ylethynyl) 3-methylaniline 2 and 2,9-bis[trans-Pt(PEt₃)₂NO₃] phenanthrene 3 gave SCC 5. Both SCCs were found to have low energy absorption and emission band maxima in dichloromethane (Table 1) (40). The low energy absorption and emission band maxima of ligand 1 were blue shifted compared with those of 4 by 57 nm (3,554 cm⁻¹) and 100 nm (4,540 cm⁻¹), respectively. A similar trend was observed for SCC 5 (Table 1). Our recent measurements of the fluorescence lifetimes of related model complexes, wherein a ligand is capped by two Pt centers, gave lifetimes on the order of 1–2 ns. These lifetimes are similar to those of the free ligands, suggesting that ligand-centered excited states are responsible for the observed emission (41).

Fig. 1.

Synthesis of organoplatinum rhomboidal SCCs 4 and 5.

Table 1.

Photophysical properties of compounds 1, 2, 4, and 5

Compound	Absorption bands λmax (nm) [ε × 10−3 (cm−1⋅M−1)]	λex (nm)	λem (nm)
1*,†	373 [13.7]	356	422
2‡,§	390 [16.7]	390	458
4*,†	317 [112], 430 [39.9]	430	522
5†,‡	318 [91.4], 432 [29.5]	430	538

^*From ref. 39.

^†Solvent: dichloromethane.

^‡From ref. 40.

^§Solvent: DMSO.

Quantum Yields of the Metallacycles and Free Ligands in an Aqueous Solution.

To confirm that the visible-light emissive properties of SCCs observed in organic solvents (Table 1) also hold true in the aqueous milieu typical for cell culture assays, we collected the absorption and emission and measured the quantum yields of the free ligands 1 and 2 and the SCCs 4 and 5 in 0.2% (vol/vol) aqueous DMSO. The results are summarized in Table 2. The free ligands have low quantum yields of 0.7%, which increases 13-fold for SCC 4 (Φ = 9.1%) and 7-fold for SCC 5 (Φ = 5.8%), respectively. In organic solvent (aerated dichloromethane), a similar trend was found: SCC 4 has a quantum yield of 28% and SCC 5 of 12% (40). The absorption and emission spectra of the SCCs in 0.2% (vol/vol) aqueous DMSO are similar to those in aerated dichloromethane. These data confirm that the SCCs are soluble and stable and their emission can be observed in a biologically compatible milieu.

Table 2.

Quantum yields of compounds 1, 2, 4, and 5 in water–DMSO (0.2% vol/vol)

Compound	Φ (%)	λex (nm)	λem range (nm)
1	0.7	362	400–600
2	0.7	380	400–600
4	9.1	380	400–700
5	5.8	390	400–700

Cellular Uptake of Metallacycles.

To assess the localization of 4 and 5 within cells, it was first necessary to evaluate their cytotoxicity. A standard cell viability assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was performed for the HeLa (human cervical epithelial adenocarcinoma) and A549 (human alveolar basal epithelial adenocarcinoma) cells; ligand 2 was used as a control. After 48 h of incubation, no appreciable decrease in metabolism was observed in both cell lines (Fig. 2), suggesting low cytotoxicity of SCCs 4 and 5 and ligands 1 and 2.

Fig. 2.

Organoplatinum rhomboidal SCCs have low cytotoxicity. Data from MTT assay are shown: (A) HeLa and (B) A549 cells treated for 48 h with ligand 2 and rhomboids 4 and 5 at concentrations ranging from 1 nM to 5 µM.

The uptake and localization of ligand 2 and rhomboids 4 and 5 in live cells were studied with laser-scanning confocal microscopy (LSCM). Two cell lines were used: HeLa and A549 (described above). Cells were plated in glass-bottom microscopy dishes and after attachment, the cells were incubated with rhomboids 4 and 5 or ligand 2 (negative control) for 4 h. Upon irradiation at λ_ex = 488 nm, the emission of 4 and 5 was monitored at 514 nm and 525 nm, respectively, whereas the background emission of ligand 2 (λ_ex = 390 nm, λ_em= 458 nm) was used as a control. The confocal images of the HeLa cells showed that both SCCs rapidly localized within the cells, forming a punctate pattern, presumably due to lysosomal accumulation (SCC 4, SI Appendix, Fig. S1 A–C; SCC 5, Fig. 3 B–D). The signals of 4 and 5 were also readily detectable in the A549 cells (SCC 4, SI Appendix, Fig. S1 D–F; SCC 5, Fig. 3 F–H). Furthermore, we found that 4 and 5 are photochemically stable inside the cells, with no appreciable photobleaching. As expected, upon excitation at λ_ex = 488 nm, the emission of ligand 2 in the λ_em = 514-nm to 525-nm range was below the detection limit of the confocal instrument (Fig. 3 A and E).

Fig. 3.

LSCM images of SCCs localized in live cells. (A) Overlay of confocal and differential interference contrast (DIC) images of HeLa cells treated with ligand 2. (B–D) Images of HeLa cells treated with SCC 5: B, confocal image; D, DIC image; and C, overlay of the signals from the fields B and D. (E) overlay of confocal DIC images of A549 cells treated with ligand 2. (F–H) A549 cells treated with SCC 5: F, confocal image; H, DIC image; and G, overlay of the signals from the fields F and H. (Scale bars: 10 µm.)

Stability of the Metallacycles in Cell Culture.

SCCs are assembled under thermodynamic control through multiple kinetically labile platinum–ligand interactions, and hence, the concern remains of how long they remain intact within cells. A preliminary assessment of in vitro stability within cells was performed, wherein the cells were treated with SCCs 4 and 5 (1.25 µM final concentration) and LSCM imaging was performed under the conditions of excitation of the free ligand. In a parallel setup, the cells were treated with free ligands 1 and 2 (5 µM final concentration) and the emission of the free ligand was quantified and normalized, and its intensity was compared with the emission of the free ligand in cells treated with SCCs 4 and 5. Ligands 1 and 2 have their absorbance maxima in the UV region (365–390 nm), and hence, a two-photon excitation mode with λ_ex = 790 nm was used. As shown in Fig. 4 A–C, free ligand 2 is uniformly distributed and readily detectable in the cytoplasm on the A549 cells. A similar distribution was observed for ligand 1 in A549 cells (SI Appendix, Fig. S3 A–C). The emission of the free ligands was normalized and compared with the emission of the ligands in the cells treated with SCCs 4 and 5. For example, when SCC 5 was imaged under the identical LSCM setup conditions, the emission at λ_em of the assembly was readily detectable but the free ligand fluorescence was low (Fig. 4E). The overlay images reveal that the signals of SCC 5 and free ligand 2 often do not colocalize (Fig. 4F), suggesting different trafficking properties of these compounds. Similarly, in HeLa cells the signals for free ligands 1 (SI Appendix, Fig. S4 A–C) and 2 (SI Appendix, Fig. S5 A–C) are clearly observable. When HeLa cells are treated with the SCCs, the signals of the free ligands are detectable and also often do not colocalize with the signals of the assemblies (SI Appendix, Figs. S4 D–G and S5 D–G). The partial overlap of the absorption spectra of the free ligands and the SCCs prevented us from quantifying and accurately comparing their signal intensities. However, these data suggest that under our confocal microscopy conditions the endocytosed SCCs at least in part remain intact.

Fig. 4.

(A–C) LSCM images of A549 cells treated with ligand 2, using excitation and emission wavelengths of the ligand in a two-photon mode (A, confocal image; B, DIC image; C, overlay of the signals from the fields A and B). (D–F) LSCM images of A549 cells treated with SCC 5, using excitation and emission wavelengths of the SCC assembly 5 or free ligand 2 (D, confocal image obtained at excitation wavelength of SCC 5; E, confocal image obtained at excitation wavelength of ligand 2; F, an overlay of the signals from the fields D and E). (Scale bar: 10 µm.)

In Vivo Antitumor Activity of the Metallacycles.

We investigated the effect of SCC 4 on the tumor growth rate in a breast cancer mouse xenograft model. Due to the lack of systemic toxicity in cell culture, we opted for not conducting the maximum tolerated dose-finding study at this time. Mouse tumor xenografts were established, by injecting immunocompromised nude mice with MDA-MB-231 cells. The tumor-bearing mice were randomly assigned to treated and control groups when the tumor volumes reached 200 mm³. The solubility of SCC 4 is limited to 0.6 mg/mL in PBS:DMSO = 1:1 (vol/vol), which translates into a maximum dose of 6 mg/kg (300 µL per average animal, weighing 30 g). The treated groups then received i.p. injections of 6 mg/kg SCC 4 in PBS:DMSO (1:1 vol/vol), whereas the control groups received injections of PBS:DMSO (1:1 vol/vol) of 300 μL per animal. Tumor sizes were monitored daily for both groups (Fig. 5 A and B). No obvious signs of toxicity were observed, as assessed by daily weight measurements and visual inspections of the appearance and the behavior of the treated mice. The treated and control mice maintained their weights at 105 ± 3% before euthanasia (Fig. 5C), suggesting low toxicity of SCCs. The median tumor volume was smaller (210 mm³) in the treated group compared with the control group (400 mm³), indicating 88% median tumor volume reduction (P < 0.001), calculated throughout the entire duration of the experiment. Notably, we achieved the tumor growth inhibition (T/C %) value of 36% at the last day of the experiment (Fig. 5D). T/C % is defined as the ratio of the median tumor volume for the treated vs. the control group at a particular day. A smaller value of T/C % ratio reflects better tumor growth inhibition. According to the National Cancer Institute, a T/C <42% (43) is a standard as a lower threshold for the efficacy of an anticancer compound.

Fig. 5.

Effect of SCC 4 treatment on tumor growth rate in MDA-MB-231 xenografts. (A) Box-and-whisker plots of the percentages of tumor volumes measured throughout the duration of the experiment. Boxes represent the upper and lower quartiles and the median, and the error bars show maximum and minimum tumor volumes. (B and C) Tumor volume (B) and weight measurements (C) of control (–O–) and SCC 4-treated (–■–) mice engrafted with MDA-MB-231 tumors through the course of the study. Error bars are ±SEM of the corresponding measurements of the mice within each experimental group. (D) Representative pictures of tumors, excised from control and SCC 4-treated mice. (E) Localization of the near-infrared contrast agent IR-783 in the tumors of the control and treated mice. The signal was processed with Living Image software with one representative sample for each group presented above. Mice from the SCC 4-treated group show lower intensity of the signal originating from the tumor-accumulated contrast agent compared with the control group.

At the endpoint of the experiment, the mice were injected with near-infrared (NIR) tumor-targeting contrast agent IR-783 and imaged using the Xenogen IVIS 200 system. The intensity of the NIR signal in the SCC 4-treated mice was consistently lower than that in vehicle-treated mice (Fig. 5E). Next, tumors from control and treated mice were excised (Fig. 5D), dissected, and used for histopathology studies. We used a hematoxylin and eosin (H&E) stain to evaluate cell morphology (Fig. 6A) in the tissue from the treated and control groups. The cells in the treated tumors appear considerably more differentiated with the greater cytoplasm-to-nucleus ratios. Cell proliferation marker Ki-67 was also used to determine the pattern of proliferating cells. Tumors in the untreated mice exhibit a 2.4-fold higher cell proliferation (Fig. 6B), as assessed by quantification of the images with Ki-67 stain (Fig. 6C) (44).

Fig. 6.

(A) Hematoxylin and eosin (H&E)-stained sections of MDA-MB-231 xenografts (purple, nuclei; pink, cytoplasm) from the control and treated with SCC 4 mice. (B) Anti-Ki-67–stained MDA-MB-231 xenografts (brown stain), from the control and treated with SCC 4 mice. (Scale bars: 50 μm.) (C) Quantification of Ki-67–stained images, using ImageJ with the ImmunoRatio plugin (44). ***P < 0.001, t test.

In summary, we report for the first time to our knowledge that emissive, rhomboidal Pt(II)-based SCCs are taken up by tumor cells and exhibit antitumor activity. The SCCs were water soluble in the physiological range of concentrations and nontoxic to cells. They remain intact upon cellular internalization and did not photobleach under the conditions of the confocal microscopy experiment. In mouse tumor xenograft models of breast cancer MDA-MB-231, treatment with SCC 4 resulted in a substantial 64% reduction of the average tumor burden on the last day of the experiment. The well-defined geometry, presence of an internal cavity, and ability to emit within the visible spectrum make these endohedral amine-functionalized SCCs attractive candidates for further development as anticancer agents. Furthermore, the emissive properties open an intriguing possibility for future development of these SCCS as agents in image-guided drug delivery (45, 46).

Methods

Quantum Yields.

Absorption spectra were recorded on a Beckman Coulter DU 800 spectrophotometer, and emission spectra were recorded on a Horiba Jobin Yvon FluoroLog spectrofluorometer using 1-cm quartz cuvettes. All samples were freshly prepared for each measurement from DMSO stock to yield solutions in water with 0.2% DMSO. The instrument was cross-calibrated with 5-carboxyfluorescein in 10 mM sodium phosphate buffer at pH 9.4 and rhodamine 6G in ethanol at excitation wavelengths of 488 nm with Φ = 98% and 480 nm with Φ = 90%, respectively. The experimental quantum yields were calculated using both standards, and the resultant values were averaged.

Cell Lines.

Human cervical epithelial carcinoma (HeLa) and human alveolar basal epithelial adenocarcinoma (A549) cell lines were obtained from ATCC. Human breast epithelial adenocarcinoma cells stably transfected with a hypoxia response element (HRE) luciferase construct and neomycin resistant gene (MDA-MB-231) was a gift of Dr. Robert Gillies, University of Arizona, Tucson AZ.

Cell Culture.

HeLa cells were grown in high glucose dulbecco's modified eagle's medium (DMEM, Invitrogen) supplemented with 10% (vol/vol) FBS (Irvine Scientific), 50 units/mL penicillin and 50 µg/mL streptomycin (Pen-Strep, Invitrogen). A549 cells were grown in RPMI 1640 (Invitrogen) supplemented with 10% or 1% (vol/vol) FBS, 50 units/mL penicillin and 50 µg/mL streptomycin. MDA-MB-231 cells were grown in DMEM supplemented with 10% (vol/vol) FBS, and 0.4 g/L geneticin. All cells were incubated at 37 ^oC in a humidified atmosphere with 5% CO₂. Cell growth and morphology were monitored by bright field microscopy. Cells were detached using trypsin in PBS (0.05%, Invitrogen).

Cell Viability Assays.

HeLa cells were seeded in a 96-well plate at a density of 5,000 cells/well in 200 µl of medium per well with 10% (vol/vol) FBS and allowed to form a monolayer for 72 h. Next, the medium was replaced with 150 µL of fresh medium containing 10% (vol/vol) FBS, 2, 4, or 5 at a concentration range from 0.001 µM to 5 µM (here and below the concentration of 4 and 5 is represented as concentration of Pt in these compounds) and 0.1% dimethyl sulfoxide (DMSO). After 48 h of incubation with compounds, 17 µL of 5 mg/mL in PBS solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) was added to each well and the plates were incubated at 37 °C and 5% CO₂ for additional 3 h. After that, the medium was carefully removed and purple formazan crystals were dissolved in DMSO (100 µL per well). The absorbance was measured at 570 nm with a correction at 690 nm in order to quantify the amount of formazan. All experiments were performed in a quadruplicate.

A549 cells were added to a 96-well plate at a density of 10,000 cells/well in 200 µl per well of medium with 10% (vol/vol) FBS and allowed to form a monolayer for 48 h. After that, the medium was replaced with 150 µL of fresh medium containing 1% FBS, 2, 4, or 5 at a concentration range from 0.001 µM to 5 µM and 0.1% DMSO. After 48 h of incubation with compounds, 17 µL of 5 mg/mL in PBS solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) was added to each well and the plates were incubated at 37 °C and 5% CO₂ for additional 3 h. After that, the medium was carefully removed and purple formazan crystals were dissolved in DMSO (100 µL per well). The absorbance was measured at 570 nm with a correction at 690 nm in order to quantify the amount of formazan. All experiments were performed in a quadruplicate.

Confocal Microscopy.

HeLa and A549 cells were seeded in glass bottom microscopy dishes (MatTek) at a density of 30,000 cells/dish in 400 µl of medium supplemented with 10% (vol/vol) FBS per dish and allowed to form a monolayer for 20 h. The medium was replaced with 400 µL of fresh medium containing 10% (vol/vol) FBS, 2, 4, or 5 at a concentration of 5 µM (in Pt) and 0.1% DMSO and the dishes were further incubated at 37 °C and 5% CO₂ for additional 4 h. Imaging was performed on a Zeiss LSM 510 inverted laser-scanning confocal microscope equipped with a ×63 oil-immersion objective lens. For SCCs 4 and 5, excitation wavelength was λ_ex = 488 nm and emission λ_em = 514 nm and 525 nm, respectively. For ligands 1 and 2, a 2-photon excitation mode was used, with λ_ex = 790 nm, λ_em = 488 nm.

Live Animal Imaging.

At the experimental endpoint of the in vivo efficacy study (SI Appendix), mice were injected intraperitoneally with the tumor-targeting near-infrared dye IR-783 and imaged using Xenogen IVIS 200 small animal imager. Euthanasia was performed as recommended by the American Veterinary Panel (AVMA 202229-249, 1993). Tumors and organs (liver, kidneys, heart, and lungs) were collected and stored in zinc formalin fixative (Sigma). Tumors were examined in a histopathology study.

Immunohistochemistry.

Tumor tissues were excised and fixed with 10% formalin, embedded in paraffin, and sectioned using a standard histological procedure. For overall morphological observations, the tissue sections were stained with hematoxylin and eosin (H&E). For Ki-67 staining, paraffin sections were deparaffinized in xylene and hydrated in a decreasing concentrations of aqueous ethanol. The slides were immersed in 3% hydrogen peroxide (Sigma) for 20 min to block endogenous peroxidase activity and then washed in PBS. For antigen retrieval, the slides were placed in preheated working solution of Retrievagen A (BD Pharmingen) and heated in a steamer for 70 min. After cooling for 20 min at room temperature, slides were rinsed with PBS, treated with 10% (vol/vol) normal FBS for 30 min, and then incubated with anti-Ki-67 antibodies for 2 h. Slides were then washed with PBS and incubated with the HRP-labeled goat anti-rabbit IgG antibodies for 1 h at room temperature. After washing with PBS, a streptavidin-HRP (BD Pharmingen) was added and incubated for 30 min. Slides were then stained with 3,3'-diaminobenzidine (Vector Laboratories) for 3 min. Counterstaining was performed with hematoxylin (Vector Laboratories). After washing with distilled water, the slides were dehydrated in increasing grades of ethanol, cleared with xylene, and mounted using permanent mounting medium (Vector Laboratories). The proliferation index was determined by measuring the percentage of Ki-67 positive cells. A total of 16 randomly selected fields at 20× objective magnification from the tumors of each treatment group were examined. The pictures were quantified with ImmunoRatio plugin for ImageJ (44). The data were plotted as a mean ± SEM and analyzed for significance using the unpaired two-tailed t test.

Other Methods.

All experiments involving live animals were approved by the University of Southern California Institutional Care and Use Committee (USC IACUC), protocol #20081. Supporting figures and in vivo efficacy testing of SCC 4 are described in SI Appendix.