Tumor-specific Ligand Research Articles

Complex Structured Fluorescent Polythiophene Graft Copolymer as a Versatile Tool for Imaging, Targeted Delivery of Paclitaxel, and Radiotherapy.

Advances in polymer chemistry resulted in substantial interest to utilize their diverse intrinsic advantages for biomedical research. Especially, studies on drug delivery for tumors have increased to a great extent. In this study, a novel fluorescent graft copolymer has been modified by a drug and targeting moiety and the resulting structure has been characterized by alterations in fluorescent intensity. The polythiophene based hybrid graft copolymer was synthesized by successive organic reactions and combination of in situ N-carboxy anhydride (NCA) ring opening and Suzuki coupling polymerization processes. Initially, targeted delivery of the graft copolymer was investigated by introducing a tumor specific ligand, anti-HER2/neu antibody, on the structure. The functionalized polymer was able to differentially indicate HER2-expressing A549 human lung carcinoma cells, whereas no signal was obtained for Vero, monkey kidney epithelial cells, and HeLa, human cervix adenocarcinoma cells. After integrating paclitaxel into the structure, cell viability, cell cycle progression, and radiosensitivity studies demonstrate HER2/neu targeting polymers were most effective to inhibit cell proliferation. Importantly, the graft copolymer used had no cytotoxic effects to cells, as evidenced by cell viability and cell cycle analysis. This work clearly confirms that a specially designed and fabricated graft copolymer with a highly complex structure is a promising theranostic agent capable of targeting tumor cells for diagnostic and therapeutic purposes.

A safe, simple and efficient doxorubicin prodrug hybrid micelle for overcoming tumor multidrug resistance and targeting delivery

A pH-sensitive prodrug, TPGS-CHN-DOX, was introduced by conjugating anticancer drug, doxorubicin (DOX), onto d-α-tocopherol polyethylene glycol 1000 succinate (TPGS) via a cleavable Schiff base linkage. The prodrug was mixed with a PEGylated lipid to form a simple but multifunctional hybrid micelle system, which can realize high drug loading capability and biocompatibility, extended blood circulation time, inhibited drug resistance in cancer cells, improved therapeutic response, reduced side effects, and easy functionalities for targeting delivery. The hybrid micelles exhibited in vitro pH-sensitive drug release, enhanced cellular uptake and strengthened cytotoxicity on both drug-sensitive human breast cancer MCF-7 and resistant MCF-7/ADR cells. P-glycoprotein functional inhibition and mitochondria-associated cell apoptosis induced by TPGS were thought to play an important role in overcoming the multidrug resistance. As a result, the hybrid micelles demonstrated good anticancer efficacy in MCF-7/ADR xenograft model. Additionally, after modifying with a tumor-specific targeting peptic ligand, cRGD, the tumor growth/metastasis inhibition was further evidenced in integrin receptor overexpressed melanoma cancer B16F10 and even murine hepatocarcinoma H22 models. This TPGS-based pH-sensitive prodrug provides a safe and “Molecular economical” way in the rational design of prodrugs for overcoming multidrug resistance and targeting delivery, which can improve the potency for clinical use.

Toll-like receptor ligands in conjunction with agonist anti-OX40 mAb immunotherapy possess differential capacity to revive anergic self-reactive CD8 T cells

Abstract Previous work has shown that the induction of CD8 T cell anergy to a prostate-specific self-antigen can be reversed following ligation of the TNFR family member OX40 (CD134) with an agonist anti-OX40 mAb plus vaccination. The provision of the Toll-like receptor 4 (TLR4) ligand bacterial lipopolysaccharide (LPS) further enhanced T cell responses following combined anti-OX40/vaccine immunotherapy. Although this approach facilitated increased CD8 T cell infiltration into the prostate, these cells were unable to elicit significant destruction of the prostate tissue. In the current study, we examined the extent to which co-administration of select TLR ligands would affect the ability of anti-OX40/vaccine therapy to revive prostate-specific CD8 T cells rendered anergic in vivo. These data revealed a differential capability of TLR agonists in conjunction with anti-OX40/vaccine immunotherapy to generate robust effector CD8 T cells capable of trafficking into normal prostate epithelia and inducing tissue-specific damage. These data provide the framework for translational studies to evaluate the efficacy of administering OX40 agonists with tumor-specific vaccination and TLR ligand adjuvants to maximize T cell-mediated therapeutic responses.

Targeted drug delivery to circulating tumor cells via platelet membrane-functionalized particles

Circulating tumor cells (CTCs) are responsible for metastases in distant organs via hematogenous dissemination. Fundamental studies in the past decade have suggested that neutralization of CTCs in circulation could represent an effective strategy to prevent metastasis. Current paradigms of targeted drug delivery into a solid tumor largely fall into two main categories: unique cancer markers (e.g. overexpression of surface receptors) and tumor-specific microenvironment (e.g. low pH, hypoxia, etc.). While relying on a surface receptor to target CTCs can be greatly challenged by cancer heterogeneity, targeting of tumor microenvironments has the advantage of recognizing a broader spectrum of cancer cells regardless of genetic differences or tumor types. The blood circulation, however, where CTCs transit through, lacks the same tumor microenvironment as that found in a solid tumor. In this study, a unique “microenvironment” was confirmed upon introduction of cancer cells of different types into circulation where activated platelets and fibrin were physically associated with blood-borne cancer cells. Inspired by this observation, synthetic silica particles were functionalized with activated platelet membrane along with surface conjugation of tumor-specific apoptosis-inducing ligand cytokine, TRAIL. Biomimetic synthetic particles incorporated into CTC-associated micro-thrombi in lung vasculature and dramatically decreased lung metastases in a mouse breast cancer metastasis model. Our results demonstrate a “Trojan Horse” strategy of neutralizing CTCs to attenuate metastasis.

Abstract 4047: CD8+T cell-mediated tumor-specific induction of the NKG2D ligands to trigger NKG2D-dependent tumor cell death in vivo

Abstract Introduction: High profile studies have shown that restoring NKG2D ligand expression greatly promotes antitumor immune surveillance and tumor regression, but malignant tumor cells often down-regulate NKG2D ligands to escape from immune surveillance. Previous studies showed that engineering tumor cells with NKG2D ligand resulted in inhibition of tumor progression. However, it is not feasible to engineer each tumor cell with NKG2D ligand in clinical treatment. Therefore, a therapeutic treatment that can restore NKG2D ligand on tumor cells is required to promote tumor immune surveillance. In this regard, many therapeutic approaches were discovered to induce NKG2D ligands on tumor cells in vitro. Unfortunately, no effective in vivo approaches are available to restore this ligand across different types of tumors because the classic in vitro stress signal-dependent NKG2D ligand induction has rarely been duplicated in solid tumors in vivo. Experimental procedure: This study reveals a unique approach_two independent co-administration of immune stimulatory signal interleukin-12 and doxorubicin_restores the NKG2D ligands in multiple murine and human tumor types. The induction of NKG2D ligands was determined using immunoblotting, flow cytometry and immunohistochemistry staining. The comparison of Rae-1 induction in immune competent mice versus immune deficient mice (nude, Rag2 -/- or Pfp -/-) implied that mature CD8+T cells are the most critical immune subpopulation. The concentration of interferon γ in tumors was measured using ELISA assay. The accumulation of NKG2D positive immune cells in tumor sections was examined using immunofluorescence straining, followed by TUNEL assay to determine tumor cell death. Results: We showed for the first time that a chemoimmunotherapy led to the tumor specific restoration of NKG2D ligands in a variety of tumor models in vivo. Systemic delivery of interleukin-12 gene therapy plus doxorubicin effectively restored murine NKG2D ligand Rae-1 in vivo in two subcutaneous tumor models (LLC and CT26), one orthotopic tumor model (K7M3) and one spontaneous breast carcinoma model. Significantly, such therapeutic approach can also stimulate human NKG2D ligands in xenograft tumor models. Intriguingly, this tumor-specific Rae-1 induction in vivo requires CD8+T cells. This NKG2D ligand-restoring therapy itself can also attract endogenous NKG2D-positive cells to attack tumor cells. Along with the tumor-specific NKG2D ligand induction, the promoted immune surveillance leads to massive tumor cell death. Conclusion: This study provides the first therapeutic treatment for inducing immune surveillance ligand in tumors in vivo. Further, it reveals a CD8+T cell-dependent novel mechanism for the restoration of NKG2D ligand. The revealed in vivo NKG2D ligand restoring approach could be translated for cancer prevention and treatment. Citation Format: Jiemiao Hu, Xueqing Xia, Eugenie S. Kleinerman, Shulin Li. CD8+T cell-mediated tumor-specific induction of the NKG2D ligands to trigger NKG2D-dependent tumor cell death in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4047. doi:10.1158/1538-7445.AM2015-4047

Folate Receptor–Positive Circulating Tumor Cell Detected by LT-PCR–Based Method as a Diagnostic Biomarker for Non–Small-Cell Lung Cancer

To investigate the diagnostic performance of folate receptor-positive circulating tumor cells in distinguishing non-small-cell lung cancer (NSCLC) from lung benign disease by using a novel ligand-targeted polymerase chain reaction (PCR) detection technique. Circulating tumor cells were enriched from 3-ml peripheral blood by immunomagnetic depletion of leukocytes and then labeled with a conjugate of a tumor-specific ligand folic acid and a synthesized oligonucleotide. After washing off free conjugates, the stripped bound conjugates were analyzed by quantitative PCR. Seven hundred fifty-six participants (473 patients with NSCLC, 227 patients with lung benign disease, and 56 healthy donors) were randomly assigned to a training set and a test set. The circulating tumor cell (CTC) levels in patients with NSCLC were significant higher than those with lung benign disease (p < 0.001) and healthy donors (p < 0.001). Compared with carcinoembryonic antigen, neuron-specific enolase, and Cyfra21-1, CTCs displayed the highest area under the receiver operating characteristic curve (training set, 0.815; validation set, 0.813) in the diagnosis of NSCLC, with a markedly sensitivity (training set, 72.46%; validation set, 76.37%) and specificity (training set, 88.65%; validation set, 82.39%). The model combining CTCs with carcinoembryonic antigen, neuron-specific enolase, and Cyfra21-1 was more effective for the diagnosis of NSCLC than tumor makers alone (sensitivity and specificity in the training set, 84.21% and 83.91%; validation set, 88.78% and 87.36%, respectively). In addition, the CTC levels were higher in patients with stage III/IV NSCLC compared with those with stage I/II disease. Ligand-targeted PCR technique was feasible and reliable for detecting folate receptor-positive CTCs in patients with NSCLC, and CTC levels could be used as a useful biomarker for the diagnosis of NSCLC.

Quantitation of rare circulating tumor cells by folate receptor α ligand-targeted PCR in bladder transitional cell carcinoma and its potential diagnostic significance.

Numerous attempts for detection of circulating tumor cells (CTC) have been made to develop reliable assays for early diagnosis of cancers. In this study, we validated the application of folate receptor α (FRα) as the tumor marker to detect CTC through tumor-specific ligand PCR (LT-PCR) and assessed its utility for diagnosis of bladder transitional cell carcinoma (TCC). Immunohistochemistry for FRα was performed on ten bladder TCC tissues. Enzyme-linked immunosorbent assay (ELISA) for FRα was performed on both urine and serum specimens from bladder TCC patients (n = 64 and n = 20, respectively) and healthy volunteers (n = 20 and n = 23, respectively). Western blot analysis and qRT-PCR were performed to confirm the expression of FRα in bladder TCC cells. CTC values in 3-mL peripheral blood were measured in 57 bladder TCC patients, 48 healthy volunteers, and 15 subjects with benign urologic pathologies by the folate receptor α ligand-targeted PCR. We found that FRα protein was overexpressed in both bladder TCC cells and tissues. The levels of FRα mRNA were also much higher in bladder cancer cell lines 5637 and SW780 than those of leukocyte. Values of FRα were higher in both serum and urine specimens of bladder TCC patients than those of control. CTC values were also higher in 3-mL peripheral blood of bladder TCC patients than those of control (median 26.5 Cu/3 mL vs 14.0 Cu/3 mL). Area under the receiver operating characteristic (ROC) curve for bladder TCC detection was 0.819, 95 % CI (0.738-0.883). At the cutoff value of 15.43 Cu/3 mL, the sensitivity and the specificity for detecting bladder cancer are 82.14 and 61.9 %, respectively. We concluded that quantitation of CTCs through FRα ligand-PCR could be a promising method for noninvasive diagnosis of bladder TCC.

Quantification of Rare Circulating Tumor Cells in Non-Small Cell Lung Cancer by Ligand-Targeted PCR

BackgroundQuantification of circulating tumor cells (CTC) is valuable for evaluation of non-small cell lung cancer (NSCLC). The sensitivity of current methods constrains their use to detect rare CTCs in early stage. Here we evaluate a novel method, ligand-targeted polymerase chain reaction (LT-PCR), that can detect rare CTCs in NSCLC patients.MethodsCTCs were enriched by immunomagnetic depletion of leukocytes and then labeled by a conjugate of a tumor-specific ligand and an oligonucleotide. After washing off free conjugates, the bound conjugates were stripped from CTCs and then analyzed by qPCR. To evaluate the clinical utility, blood samples were obtained from 72 NSCLC patients (33 initially diagnosed and 39 on chemotherapy), 20 benign patients, and 24 healthy donors.ResultsExperiments with healthy blood spiked with tumor cells indicated the LT-PCR allows specific detection of CTC. The clinical study showed that the initially diagnosed patients have an average of 20.8 CTC units with metastatic diseases, 11.8 CTC units with localized diseases, and 6.0 CTC units with benign diseases. With the threshold of 8.5 CTC units, the assay can detect 80% of stage I/II, 67% of stage III, and 93% of stage IV cancer. With the benign patients and healthy donors as control group, the method can detect cancer with a sensitivity of 81.8% and a specificity of 93.2%.ConclusionThe LT-PCR would allow quantification of CTC in NSCLC patients at a more sensitive level, providing a potential tool for stratifying malignant lung diseases, especially at early stage.

Folate Receptor-Positive Circulating Tumor Cells as a Novel Diagnostic Biomarker in Non-Small Cell Lung Cancer

The study aims to determine the efficacy and feasibility of a novel folate receptor (FR)-based circulating tumor cell (CTC) detection method in the diagnosis of non-small cell lung cancer (NSCLC). CTCs were collected from 3 ml of blood based on negative enrichment by immunomagnetic beads and then labeled by a conjugate of a tumor-specific ligand folate and an oligonucleotide. After washing off redundant conjugates, the bound conjugates were removed and analyzed by quantitative polymerase chain reaction. The captured cells were validated as tumor cells by immunofluorescence staining. In the evaluation of clinical utility, the results showed that the CTC levels of 153 patients with NSCLC were significantly higher than the controls (49 healthy donors and 64 patients with benign lung diseases; P < .001). With a threshold of 8.64 CTC units, the method showed a sensitivity of 73.2% and a specificity of 84.1% in the diagnosis of NSCLC, especially a sensitivity of 67.2% in stage I disease. Compared with the existing clinical biomarkers such as neuron-specific enolase (NSE), carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), cyfra21-1, and squamous cell carcinoma antigen (SCC Ag), the method showed the highest diagnostic efficiency (area under the curve, 0.823; 95% confidence interval, 0.773-0.874). Together, our results demonstrated that FR-positive CTCs were feasible diagnostic biomarkers in patients with NSCLC, as well as in early-stage tumors.

Folate-conjugated cross-linked magnetic nanoparticles as potential magnetic resonance probes for in vivo cancer imaging.

Superparamagnetic iron oxide nanoparticles are widely used as nanoprobes for magnetic resonance imaging (MRI). In this study, a novel type of cross-linked magnetic nanoparticle was developed in an effort to improve the structural stability of amphiphilic polymer-coated iron oxide nanoparticles. Iron oxide nanocrystals were coated with a cross-linkable amphiphilic graft copolymer, poly(succinimide) grafted with folate-conjugated polyethylene glycol (PEG) and alkyl chains. The tumor-specific targeting ligand, folate, was included to target and detect cancer cells. The hydrophobic portions of the amphiphilic copolymer on the surfaces of the nanoparticles were cross-linked via an aminolysis reaction between the succinimide units and a bifunctional primary amine. The folate-conjugated cross-linked magnetic nanoparticles (F-CLMNPs) were 40 nm in diameter and displayed a low cytotoxicity, even at relatively high concentrations. The F-CLMNPs exhibited highly efficient intracellular uptake into KB cells, which overexpress the folate receptor, as determined by fluorescence microscopy, flow cytometry, and Prussian blue staining. In vivo MR images of a mouse bearing a KB cell tumor displayed a 75% drop in the T2 signal in the tumor tissues within 3 hours. These results indicated that the F-CLMNPs accumulated at the tumor site and were highly effective for tumor detection using in vivo MRI techniques.

Doxorubicin loaded silica nanorattles actively seek tumors with improved anti-tumor effects

Silica nanorattles (SNs) have proven to be promising vehicles for drug delivery. In order to further enhance efficacy and minimize adverse effects, active targeted delivery to tumors is necessary. In this work, SNs modified with a tumor specific targeting ligand, folic acid (FA), was used as carrier of doxorubicin (DOX) (DOX-FA-SNs). Drug loading, cytotoxicity and cellular uptake of DOX-FA-SNs in vitro in human cervical carcinoma cells (HeLa cells) were evaluated. DOX-FA-SNs showed a higher cytotoxicity in human cervical carcinoma cells (HeLa cells) than DOX loaded carboxyl (-COOH) and poly(ethylene glycol) (PEG) modified SNs (DOX-COOH-SNs and DOX-PEG-SNs, respectively). However, DOX-FA-SNs showed lower cytotoxicity in folate receptor negative normal mouse fibroblast cells (L929 cells) compared with free DOX. In vivo tumor-targeted fluorescence imaging indicated specific tumor targeting and uptake of FA-SNs in nude mice bearing subcutaneous HeLa cell-derived xenograft tumors. In vivo anti-tumor experiments demonstrated that DOX-FA-SNs (10 mg kg(-1) of DOX) significantly regressed the tumor growth and reduced toxicity compared with free DOX. These results have great significance in developing and optimizing SNs as effective intracellular delivery and specific tumor targeting vehicles.

Chlorotoxin-modified macromolecular contrast agent for MRI tumor diagnosis

Clinical diagnosis of cancers using magnetic resonance imaging (MRI) is highly dependent on contrast agents, especially for brain tumors which contain blood–brain barrier (BBB) at the early stage. However, currently mostly used low molecular weight contrast agents such as Gd-DTPA suffer from rapid renal clearance, non-specificity, and low contrast efficiency. The aim of this paper is to investigate the potential of a macromolecular MRI contrast agent based on dendrigraft poly- l-lysines (DGLs), using chlorotoxin (CTX) as a tumor-specific ligand. The contrast agent using CTX-modified conjugate as the main scaffold and Gd-DTPA as the payload was successfully synthesized. The results of fluorescent microscopy showed that the modification of CTX could markedly enhance the cellular uptake in C6 glioma and liver tumor cell lines, but not in normal cell line. Significantly increased accumulation of CTX-modified conjugate within glioma and liver tumor was further demonstrated in tumor-bearing nude mice using in vivo imaging system. The MRI results showed that the signal enhancement of mice treated with CTX-modified contrast reached peak level at 5 min for both glioma and liver tumor, 144.97% ± 19.54% and 158.69% ± 12.41%, respectively, significantly higher than that of unmodified counterpart and commercial control. And most importantly, the signal enhancement of CTX-modified contrast agent maintained much longer compared to that of controls, which might be useful for more exact diagnosis for tumors. CTX-modified dendrimer-based conjugate might be applied as an efficient MRI contrast agent for targeted and accurate tumor diagnosis. This finding is especially important for tumors such as brain glioma which is known hard to be diagnosed due to the presence of BBB.

Tumor-specific hybrid polyhydroxybutyrate nanoparticle: Surface modification of nanoparticle by enzymatically synthesized functional block copolymer

A new approach to functionalize the surface of hydrophobic nanocarrier through enzymatic polymerization was demonstrated. The effective coupling between the hydrophobic surface of PHB nanoparticle and PHB chain grown from the enzyme fused with a specific ligand provided a simple way of functionalizing nanoparticles with active protein layers in aqueous environment. PHB nanoparticles loaded with model drug molecule, Nile red, were prepared through oil-in-water emulsion solvent evaporation method and the surface of nanoparticles were functionalized with tumor-specific ligand, RGD4C, fused with PHA synthase that drove the coupling reaction. The functionalized PHB nanoparticles showed a specific affinity to MDA-MB 231 breast cancer cells indicating that the tumor-specific ligand, RGD4C, was effectively displayed on the surface of PHB nanoparticles through enzymatic modification and confers targeting capability on the drug carrier.

PH-Sensitive siRNA Nanovector for Targeted Gene Silencing and Cytotoxic Effect in Cancer Cells

A small interfering RNA (siRNA) nanovector with dual targeting specificity and dual therapeutic effect is developed for targeted cancer imaging and therapy. The nanovector is composed of an iron oxide magnetic nanoparticle core coated with three different functional molecules: polyethyleneimine (PEI), siRNA, and chlorotoxin (CTX). The primary amine group of PEI is blocked with citraconic anhydride that is removable at acidic conditions, not only to increase its biocompatibility at physiological conditions but also to elicit a pH-sensitive cytotoxic effect in the acidic tumor microenvironment. The PEI is covalently immobilized on the nanovector via a disulfide linkage that is cleavable after cellular internalization of the nanovector. CTX as a tumor-specific targeting ligand and siRNA as a therapeutic payload are conjugated on the nanovector via a flexible and hydrophilic PEG linker for targeted gene silencing in cancer cells. With a size of ∼60 nm, the nanovector exhibits long-term stability and good magnetic property for magnetic resonance imaging. The multifunctional nanovector exhibits both significant cytotoxic and gene silencing effects at acidic pH conditions for C6 glioma cells, but not at physiological pH conditions. Our results suggest that this nanovector system could be safely used as a potential therapeutic agent for targeted treatment of glioma as well as other cancers.

Folic acid–Pluronic F127 magnetic nanoparticle clusters for combined targeting, diagnosis, and therapy applications

Superparamagnetic iron oxides possess specific magnetic properties in the presence of an external magnetic field, which make them an attractive platform as contrast agents for magnetic resonance imaging (MRI) and as carriers for drug delivery. In this study, we investigate the drug delivery and the MRI properties of folate-mediated water-soluble iron oxide incorporated into micelles. Pluronic ® F127 (PF127), which can be self-assembled into micelles upon increasing concentration or raising temperatures, is used to decorate water-soluble polyacrylic acid-bound iron oxides (PAAIO) via a chemical reaction. Next, the hydrophobic dye Nile red is encapsulated into the hydrophobic poly(propylene oxide) compartment of PF127 as a model drug and as a fluorescent agent. Upon encapsulation, PAAIO retains its superparamagnetic characteristics, and thus can be used for MR imaging. A tumor-specific targeting ligand, folic acid (FA), is conjugated onto PF127–PAAIO to produce a multifunctional superparamagnetic iron oxide, FA–PF127–PAAIO. FA–PF127–PAAIO can be simultaneously applied as a diagnostic and therapeutic agent that specifically targets cancer cells that overexpress folate receptors in their cell membranes. PF127–PAAIO is used as a reference group. Based on FTIR and UV–vis absorbance spectra, the successful synthesis of PF127–PAAIO and FA–PF127–PAAIO is realized. The magnetic nanoparticle clusters of PF127–PAAIO and FA–PF127–PAAIO are visualized by transmission electron microscope (TEM). FA–PF127–PAAIO, together with a targeting ligand, displays a higher intracellular uptake into KB cells. This result is confirmed by laser confocal scanning microscopy (CLSM), flow cytometry, and atomic absorption spectroscopy (AAS) studies. The hysteresis curves, generated by using a superconducting quantum interference device (SQUID) magnetometer analysis, demonstrate that the magnetic nanoparticles are superparamagnetic with insignificant hysteresis. The MTT assay explains the negligible cell cytotoxicity of PF127–PAAIO and FA–PF127–PAAIO. In KB cells, the in vitro MRI study indicates the better T 2-weighted images in FA–PF127–PAAIO than in PF127–PAAIO.

The use of biotinylated-EGF-modified gelatin nanoparticle carrier to enhance cisplatin accumulation in cancerous lungs via inhalation

To develop a polymer-anticancer drug conjugate, we employed gelatin nanoparticles (GPs) as carriers of cisplatin (CDDP) with anticipated improved therapeutic effect and reduced side effects. The anticancer activities of CDDP-incorporated in GPs (GP–Pt) with biotinylated-EGF (bEGF) modification (GP–Pt–bEGF) were studied. GP–Pt–bEGF with EGFR affinity produced much higher Pt concentrations in A549 cells (high EGFR expression) than in HFL1 cells (low EGFR expression). An in vitro anticancer study showed that GP–Pt–bEGF was more potent than free CDDP or GP–Pt because of its rapid effect on the cell cycle as well as a lower IC 50 (1.2 μg/ml) that inhibits A549 cell growth. PI staining showed that cells treated with GP–Pt-bEGF for only 4 h had the highest sub-G1 population. The CDDP formulations – free CDDP, GP–Pt, and GP–Pt–bEGF – were given by intratumorous injections to SCID mice in a subcutaneous model. This treatment showed that GP–Pt–bEGF had stronger anti-tumor activity and was less toxic than free CDDP in vivo. Mice treated with GP–Pt–bEGF showed slight body weight loss, whereas free CDDP treatment at the same dose caused a body weight loss of 20–30%. Furthermore, these formulations were given to mice with lung cancer via aerosol delivery. This treatment showed that inhaled GP–Pt–bEGF could target EGFR-overexpressing cells to achieve high cisplatin dosage in cancerous lungs. To summarize, gelatin nanoparticles loaded with CDDP and decorated with EGF tumor-specific ligand were successfully developed. Their in vitro and in vivo targeting ability and anticancer effect were confirmed. The aerosol delivery of the nanodrug carrier was demonstrated. Simple aerosol delivery of targeted drug carriers may prove useful for the clinical treatment of lung cancer patients.

Quantification of Diphtheria Toxin–Mediated ADP-Ribosylation in a Solid-Phase Assay

Because of reduced vaccination programs, the number of diphtheria infections has increased in the last decade. Diphtheria toxin (DT) is expressed by Corynebacterium diphtheriae and is responsible for the lethality of diphtheria. DT inhibits cellular protein synthesis by ADP-ribosylation of the eukaryotic elongation factor 2 (eEF2). No in vitro system for the quantification of DT enzymatic activity exists. We developed a solid-phase assay for the specific detection of ADP-ribosylation by DT. Solid phase-bound his-tag eEF2 is ADP-ribosylated by toxins using biotinylated NAD(+) as substrate, and the transferred biotinylated ADP-ribose is detected by streptavidin-peroxidase. DT enzymatic activity correlated with absorbance. We measured the amount of ADP-ribosylated eEF2 after precipitation with streptavidin-Sepharose. Quantification was done after Western blotting and detection with anti-his-tag antibody using an LAS-1000 System. The assay detected enzymatically active DT at 30 ng/L, equivalent to 5 mU/L ADP-ribosylating activity. Pseudomonas exotoxin A (PE) activity was also detected at 100 ng/L. We verified the assay with chimeric toxins composed of the catalytic domain of DT or PE and a tumor-specific ligand. These chimeric toxins revealed increased signals at 1000 ng/L. Heat-inactivated DT and cholera toxin that ADP-ribosylates G-proteins did not show any signal increase. The assay may be the basis for the development of a routine diagnostic assay for the detection of DT activity and highly specific inhibitors of DT.

In vivo quantitation of rare circulating tumor cells by multiphoton intravital flow cytometry

Quantitation of circulating tumor cells (CTCs) constitutes an emerging tool for the diagnosis and staging of cancer, assessment of response to therapy, and evaluation of residual disease after surgery. Unfortunately, no existing technology has the sensitivity to measure the low numbers of tumor cells (<1 CTC per ml of whole blood) that characterize minimal levels of disease. We present a method, intravital flow cytometry, that noninvasively counts rare CTCs in vivo as they flow through the peripheral vasculature. The method involves i.v. injection of a tumor-specific fluorescent ligand followed by multiphoton fluorescence imaging of superficial blood vessels to quantitate the flowing CTCs. Studies in mice with metastatic tumors demonstrate that CTCs can be quantitated weeks before metastatic disease is detected by other means. Analysis of whole blood samples from cancer patients further establishes that human CTCs can be selectively labeled and quantitated when present at approximately 2 CTCs per ml, opening opportunities for earlier assessment of metastatic disease.

APO2 ligand/tumor necrosis factor-related apoptosis-inducing ligand in prostate cancer therapy

Prostate cancer is one of the most common cancers in men and is the second leading cause of cancer-related death in the USA. Many anti-tumor agents against prostate cancer cells have been developed, but their unacceptable systemic toxicity to normal tissues usually limits their use in the clinic. Apo2 ligand (Apo2L), also called Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), is one of several members of the TNF gene superfamily that induces apoptosis through engagement of death receptors. This protein has generated tremendous enthusiasm as a potential tumor-specific cancer therapeutic because, as a stable trimer, it selectively induces apoptosis in many transformed cells, but not in most normal cells. In this review we discuss its potential use in prostate cancer therapy, the mechanisms by which induces apoptosis or that underlie resistance to it, and strategies for sensitization to overcome them. Conventional chemotherapeutic and chemopreventive drugs, irradiation, and other therapeutic agents, such as histone deacetylase inhibitors or retinoids can sensitize Apo2L/TRAIL-resistant cells and tumors. Investigating the apoptotic effects of Apo2L/TRAIL, a unique tumor-specific cell death ligand, now in clinical trials, alone or in combination may not only help in understanding its antineoplastic role in prostate carcinoma but may also provide insights into basic mechanisms of apoptosis.

529. Oncolytic Virotherapy Employing flt-1 Driven mda-7/IL-24 Gene Delivery

Top of pageAbstract Vascular-targeted anticancer therapy using conditionally replicating adenoviruses (CRAd) is an attractive strategy for cancer treatment. For specific retargeting of CRAd, selective transcription of viral genes utilizing tumor-specific promoters has been developed. CRAd targeting using insertion of the tumor-specific binding RGD ligand in the Ad fiber has shown increased infectivity. A cancer growth-suppressing gene, mda-7/IL-24, has demonstrated profound and selective anticancer activity. The central hypothesis of this study was that selective transcriptional and transductional targeting of CRAd encoding the mda-7/IL-24 gene using flt-1 promoter for specific E1a expression and RDG-4C modification of the Ad fiber-knob region can increase efficacy via direct killing of tumor endothelial cells and flt-1-positive cancer cells. Specific mda-7/IL-24 overexpression should enhance the anti-tumor efficacy of CRAd oncolytic virotherapy alone. The flt-1 promoter-driven firefly luciferase expression was determined using replication-defective Adflt-Luc and AdCMV-Luc vectors on a panel of human glioma, breast, ovarian and prostate cancer cells and human or mouse endothelial cells. The cytotoxicity of cancer and endothelial cells to CRAd infection was analyzed using the crystal violet inclusion assay. The CRAd progeny production was determined using real time PCR and a standard 50% tissue culture infectious dose assay. Tumor xenograft models were used to determinate the therapeutic efficacy of oncolytic virotherapy to treat human glioma, breast, ovarian and prostate tumors. Specificity of flt-1 promoter for human and mouse endothelial cells and U251MG glioma, PA-1 ovarian, LCC6 breast, and DU145 prostate cancer cells was demonstrated. We have developed a CRAd using the flt-1 promoter element for specific E1a gene expression, encoding the mda-7/IL-24 gene (CRAdRGDflt-mda-7) and a CDCGRDCFC-containing peptide in the HI loop of the fiber knob domain. The cancer cells demonstrated different levels of cytotoxicity after CRAdRGDflt-mda-7 infection. There was a significant decrease in viability of U251MG, PA-1, LCC6, and DU145 cells infected with CRAdRGDflt-mda-7. In vivo therapy studies demonstrated that intratumoral injection of CRAdRGDflt-mda-7 produced significant inhibition of subcutaneous LCC6 breast, OVCAR-3 ovarian, and DU145 prostate cancer xenografts and delay of D54MG human glioma tumor growth in athymic nude mice. These results suggest that selective targeting of oncolytic virotherapy with enhanced potency via mda-7/IL-24 pro-apoptotic/anti-angiogenic gene expression would be a superior strategy for treatment of human cancer.