Purpose The objective of this study was to determine local doxorubicin levels surrounding radiopaque drug-eluting beads (DEB) in normal swine liver and kidney following transcatheter arterial chemoembolization (TACE). The influence of bead size (70–150µm or 100–300µm) was compared with regard to tissue penetration and spatial distribution of the bead as well as eventual drug coverage (i.e., amount of tissue exposed to drug). Materials and Methods Radiopaque DEBs were synthesized by suspension polymerization followed by incorporation of iodized oil and doxorubicin. Chemoembolization of swine liver and kidney was performed under fluoroscopic guidance. Three dimensional tissue penetration of image-able DEB was investigated ex vivo with microCT. Drug penetration from the bead surface and drug coverage was evaluated with epi-fluorescence microscopy while cellular localization of doxorubicin was evaluated with confocal microscopy. Necrosis was evaluated with H&E. Results MicroCT demonstrated that 70–150µm DEB were present in more distal arteries and located in a more frequent and homogeneous spatial distribution. Tissue penetration of doxorubicin from the bead appeared similar (~300µm) for both DEBs with a maximum tissue drug concentration at 1hr coinciding with nuclear localization of doxorubicin. The greater spatial frequency of the 70–150µm DEBs resulted in ~2-fold improved drug coverage in kidney. Cellular death is predominantly observed around the DEBs beginning at 8 hr but increased at 24 and 168 hrs. Conclusions Smaller DEBs penetrated further into targeted tissue (macroscopic) with a higher spatial density, resulting in greater and more uniform drug coverage (microscopic) in swine.

The Asn-Gly-Arg (NGR) motif in both cyclic and linear form has previously been shown to specifically bind to CD13/aminopeptidase N that is selectively overexpressed in tumor vasculature and some tumor cells. However, previous versions of cyclic NGR used a liable disulfide bridge between cysteine residues that may be problematic for liposome targeting due to disulfide bond formation between adjacent peptides on the liposomal surface. In this study, we report the design, synthesis, and characterization of a novel cyclic NGR containing peptide, cKNGRE, which does not contain a disulfide bridge. cKNGRE was synthesized in good yield and purity and attached to the fluorescent reporter Oregon Green (cKNGRE-OG) and lysolipid-containing temperature sensitive liposome (LTSLs). The identity of cKNGRE was verified with NMR and mass spectral techniques. In vitro fluorescence microscopy evaluation of cKNGRE-OG demonstrated binding and active uptake by CD13 + cancer cells and minimal binding to CD13 − cancer cells. The cKNGRE-OG ligand displayed 3.6-fold greater affinity for CD13 + cancer cells than a linear NGR containing peptide. Affinity for CD13 + cancer cells was similarly improved 10-fold for both the cyclic and linear NGR when presented in a multivalent fashion on the surface of an LTSL. cKNGRE-targeted LTSLs rapidly released (>75% in <4 sec) doxorubicin at 41.3°C with minimal release at 37°C. These results demonstrate the ability to synthesize a cKNGRE-targeted temperature sensitive liposome that lacks a disulfide bridge and has sufficient binding affinity for biological applications.

Centrosome amplification is a pivotal mechanism underlying tumorigenesis but its role in gliomas is underinvestigated. The present study specifically examines the expression and distribution of the centrosome-associated cytoskeletal protein gamma-tubulin in 56 primary diffuse astrocytic gliomas (grades II-IV) and in 4 human glioblastoma cell lines (U87MG, U118MG, U138MG, and T98G). Monoclonal anti-peptide antibodies recognizing epitopes in C-terminal or N-terminal domains of the gamma-tubulin molecule were used in immunohistochemical, immunofluorescence, and immunoblotting studies. In tumors in adults (n = 46), varying degrees of localization were detected in all tumor grades, but immunoreactivity was significantly increased in high-grade anaplastic astrocytomas and glioblastomas multiforme as compared to low-grade diffuse astrocytomas (p = 0.0001). A similar trend was noted in diffuse gliomas in children but the sample of cases was too small as to be statistically meaningful. Two overlapping patterns of ectopic cellular localization were identified in both primary tumors and glioblastoma cell lines: A punctate pattern, in which gamma-tubulin was partially co-distributed with pericentrin in the pericentriolar region, and a diffuse pattern, independent of pericentrin staining, denoting a soluble pool of gamma-tubulin. Cellular gamma-tubulin was detected in both soluble and insoluble (nocodazole-resistant) fractions of glioblastoma cells. Divergent localizations of gamma-tubulin and pericentrin suggest a differential distribution of these 2 centrosome-associated proteins in glioblastoma cell lines. Our results indicate that overexpression and ectopic cellular distribution of gamma-tubulin in astrocytic gliomas may be significant in the context of centrosome protein amplification and may be linked to tumor progression and anaplastic potential.

We have previously shown that the neuronal-associated class III beta-tubulin isotype and the centrosome-associated gamma-tubulin are aberrantly expressed in astrocytic gliomas (Cell Motil Cytoskeleton 2003, 55:77-96; J Neuropathol Exp Neurol 2006, 65:455-467). Here we determined the expression, distribution and interaction of betaIII-tubulin and gamma-tubulin in diffuse-type astrocytic gliomas (grades II-IV) (n = 17) and the human glioblastoma cell line T98G. By immunohistochemistry and immunofluorescence microscopy, betaIII-tubulin and gamma-tubulin were co-distributed in anaplastic astrocytomas and glioblastomas and to a lesser extent, in low-grade diffuse astrocytomas (P < 0.05). In T98G glioblastoma cells betaIII-tubulin was associated with microtubules whereas gamma-tubulin exhibited striking diffuse cytoplasmic staining in addition to its expectant centrosome-associated pericentriolar distribution. Treatment with different anti-microtubule drugs revealed that betaIII-tubulin was not associated with insoluble gamma-tubulin aggregates. On the other hand, immunoprecipitation experiments unveiled that both tubulins formed complexes in soluble cytoplasmic pools, where substantial amounts of these proteins were located. We suggest that aberrant expression and interactions of betaIII-tubulin and gamma-tubulin may be linked to malignant changes in glial cells.

PurposeRadiofrequency ablation (RFA) is a minimally invasive energy delivery technique increasingly used for focal therapy to eradicate localized disease. RFA-induced tumor-cell necrosis generates an immunogenic source of tumor antigens known to induce antitumor immune responses. However, RFA-induced antitumor immunity is insufficient to control metastatic progression. We sought to characterize (a) the role of RFA dose on immunogenic modulation of tumor and generation of immune responses and (b) the potential synergy between vaccine immunotherapy and RFA aimed at local tumor control and decreased systemic progression.Experimental DesignMurine colon carcinoma cells expressing the tumor-associated (TAA) carcinoembryonic antigen (CEA) (MC38-CEA+) were studied to examine the effect of sublethal hyperthermia in vitro on the cells’ phenotype and sensitivity to CTL-mediated killing. The effect of RFA dose was investigated in vivo impacting (a) the phenotype and growth of MC38-CEA+ tumors and (b) the induction of tumor-specific immune responses. Finally, the molecular signature was evaluated as well as the potential synergy between RFA and poxviral vaccines expressing CEA and a TRIad of COstimulatory Molecules (CEA/TRICOM).Results In vitro, sublethal hyperthermia of MC38-CEA+ cells (a) increased cell-surface expression of CEA, Fas, and MHC class I molecules and (b) rendered tumor cells more susceptible to CTL-mediated lysis. In vivo, RFA induced (a) immunogenic modulation on the surface of tumor cells and (b) increased T-cell responses to CEA and additional TAAs. Combination therapy with RFA and vaccine in CEA-transgenic mice induced a synergistic increase in CD4+ T-cell immune responses to CEA and eradicated both primary CEA+ and distal CEA– s.c. tumors. Sequential administration of low-dose and high-dose RFA with vaccine decreased tumor recurrence compared to RFA alone. These studies suggest a potential clinical benefit in combining RFA with vaccine in cancer patients, and augment support for this novel translational paradigm.

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<b><i>Background:</i></b> Malignant melanoma represents the deadliest form of skin cancer with a high tendency to metastasize during the early course of the disease. Radiation therapy has long played a key role in the management of both local and metastatic melanoma. Although local radiation therapy exerts antitumor effects by damaging the cellular DNA, it also induces an important out-of-field (distant) effect known as the “abscopal effect” in nonirradiated sites. Radiation therapy-induced abscopal effects are believed to be mediated by activation and stimulation of the immune system. <b><i>Objective:</i></b> To provide a detailed overview of the current state of knowledge and clinical experience of radiation therapy-induced abscopal effects in patients with malignant melanoma. <b><i>Methods:</i></b> Using electronic databases such as MEDLINE via PubMed and Google Scholar, a systematic literature review was performed to find published clinical evidence for radiation therapy-induced abscopal effects in patients with malignant melanoma. The clinical data on radiation therapy-induced abscopal effects were reviewed and the outcomes summarized. <b><i>Results:</i></b> Clinical evidence of patients with malignant melanoma was gathered using databases from MEDLINE and those findings were summarized. Although the precise mechanism of the abscopal effect of radiation therapy is still not completely understood, evidence suggests that tumor cell destruction by radiation releases tumor antigens that stimulate the immune system of the host to activate the body’s immune effector cells systemically and produce distant non-target antitumor effects. This forms a basis for using the radiation therapy with immunotherapy to augment the abscopal response rates. <b><i>Conclusions:</i></b> Current clinical evidence suggests that there is a large potential to enhance the abscopal effect when radiation therapy is combined with immunotherapeutic agents for the treatment of malignant melanoma. Ongoing and planned clinical trials may provide us with a more in-depth understanding of how this combination therapy can be optimally utilized clinically to achieve improved survival outcomes among patients with malignant melanoma.

Objective Emerging studies show that radiation therapy produces an important out of field (distant) effect known as the ''abscopal effect^in nonirradiated tumor sites. The objective of this study was to provide an overview of the current state of knowledge and clinical experience of radiation therapy producing abscopal effects in the management of different types of malignant diseases. Methods Peer-reviewed published clinical evidence on the abscopal effect of radiation therapy was collected using electronic databases such as Medline via PubMed and Google Scholar. The reference lists were searched in the publications that we obtained in an attempt to find additional relevant publications. Non-indexed peer-reviewed journals were manually searched and relevant information was extracted. The search was restricted to English language articles. The clinical data on the abscopal effect of radiation therapy were reviewed and the outcomes have been summarized. Results Currently, only clinical case reports and anecdotes have slowly converted into solid clinical data and interest is building in the field of radiation therapy, specifically on how local radiation can produce the abscopal effects for the management of different types of malignant tumors. Extensive clinical evidence suggests that the radiation therapy induced abscopal antitumor effects are mediated by immune cells such as the T-lymphocytes. This forms a basis for using radiation therapy in combination with immunotherapy to augment the abscopal response rates in cancer patients. Current evidence demonstrates that radiation therapy induces abscopal responses across many tumor types. Conclusion Together, the clinical outcomes from published reports suggest that localized radiation therapy is capable of inducing abscopal effects in a wide variety of malignant tumors. With the advent of novel immunotherapies, the potential for immune activation by radiation defines a novel role for radiation therapy in the treatment of systemic disease. A clinical consideration of the abscopal effects produced by radiation therapy could lead to a revolutionary change in the current management of patients including radiation treatment strategies and immunotherapies for various malignant tumors.