P. Jack Hoopes

Tumor cell targeting by iron oxide nanoparticles is dominated by different factors in vitro versus in vivo.

Realizing the full potential of iron oxide nanoparticles (IONP) for cancer diagnosis and therapy requires selective tumor cell accumulation. Here, we report a systematic analysis of two key determinants for IONP homing to human breast cancers: (i) particle size and (ii) active vs passive targeting. In vitro, molecular targeting to the HER2 receptor was the dominant factor driving cancer cell association. In contrast, size was found to be the key determinant of tumor accumulation in vivo, where molecular targeting increased tumor tissue concentrations for 30 nm but not 100 nm IONP. Similar to the in vitro results, PEGylation did not influence in vivo IONP biodistribution. Thus, the results reported here indicate that the in vitro advantages of molecular targeting may not consistently extend to pre-clinical in vivo settings. These observations may have important implications for the design and clinical translation of advanced, multifunctional, IONP platforms.

Tumor Vascular Permeabilization by Vascular-Targeting Photosensitization: Effects, Mechanism, and Therapeutic Implications

Purpose: Loss of vascular barrier function has been observed shortly following vascular-targeting photodynamic therapy. However, the mechanism involved in this event is still not clear, and the therapeutic implications associated with this pathophysiologic change have not been fully explored. Experimental Design: The effect of vascular-targeting photodynamic therapy on vascular barrier function was examined in both s.c. and orthotopic MatLyLu rat prostate tumor models and endothelial cells in vitro, using photosensitizer verteporfin. Vascular permeability to macromolecules (Evans blue-albumin and high molecular weight dextran) was assessed with dye extraction (ex vivo) and intravital microscopy (in vivo) methods. Intravital microscopy was also used to monitor tumor vascular functional changes after vascular-targeting photodynamic therapy. The effects of photosensitization on monolayer endothelial cell morphology and cytoskeleton structures were studied with immunofluorescence staining. Results: Vascular-targeting photodynamic therapy induced vascular barrier dysfunction in the MatLyLu tumors. Thus, tumor uptake of macromolecules was significantly increased following photodynamic therapy treatments. In addition to vascular permeability increase, blood cell adherence to vessel wall was observed shortly after treatment, further suggesting the loss of endothelial integrity. Blood cell adhesion led to the formation of thrombi that can occlude blood vessels, causing vascular shutdown. However, viable tumor cells were often detected at tumor periphery after vascular-targeting photodynamic therapy. Endothelial cell barrier dysfunction following photodynamic therapy treatment was also observed in vitro by culturing monolayer endothelial cells on Transwell inserts. Immunofluorescence study revealed microtubule depolymerization shortly after photosensitization treatment and stress actin fiber formation thereafter. Consequently, endothelial cells were found to retract, and this endothelial morphologic change led to the formation of intercellular gaps. Conclusions: Vascular-targeting photodynamic therapy permeabilizes blood vessels through the formation of endothelial intercellular gaps, which are likely induced via endothelial cell microtubule depolymerization following vascular photosensitization. Loss of endothelial barrier function can ultimately lead to tumor vascular shutdown and has significant implications in drug transport and tumor cell metastasis.

Changes in Oxygenation of Intracranial Tumors With Carbogen: a BOLD MRI and EPR Oximetry Study

To examine, using blood oxygen level dependent (BOLD) MRI and EPR oximetry, the changes in oxygenation of intracranial tumors induced by carbogen breathing.

Assessment of cerebral pO2 by EPR oximetry in rodents: effects of anesthesia, ischemia, and breathing gas

This report describes experiments designed to assess and illustrate the effectiveness of a new method for the measurement of cerebral interstitial pO2 in conscious rodents. It is based on the use of low frequency electron paramagnetic resonance (EPR) spectroscopy with lithium phthalocyanine as the oxygen sensitive probe. Magnetic resonance imaging was used to document placement of the probe in the brain, and to assess potential cerebral changes associated with the placement. The technique provided accurate and reproducible measurements of localized pO2 in the brains of conscious rodents under a variety of physiological conditions and for time periods of at least 2 weeks. Using this approach we quantitated the depressing effects on cerebral pO2 of three representative anesthetics, isoflurane, ketamine/xylazine, and sodium pentobarbital. The effects of changing the content of oxygen in the breathing gas was investigated and found to change the cerebral pO2. In experiments with gerbils, crystals of lithium phthalocyanine were implanted in each side of the brain and using a one-dimensional magnetic field gradient, simultaneous measurement of pO2 values from normal and ischemic (ischemia induced by unilateral ligation of a carotid artery) hemispheres of the brain were obtained. These results demonstrate that EPR oximetry with lithium phthalocyanine is a versatile and useful method in the measurement of cerebral pO2 under various physiological and pathophysiological conditions.

MAGNETIC NANOPARTICLE HYPERTHERMIA IN CANCER TREATMENT

The activation of magnetic nanoparticles (mNPs) by an alternating magnetic field (AMF) is currently being explored as technique for targeted therapeutic heating of tumors. Various types of superparamagnetic and ferromagnetic particles, with different coatings and targeting agents, allow for tumor site and type specificity. Magnetic nanoparticle hyperthermia is also being studied as an adjuvant to conventional chemotherapy and radiation therapy. This review provides an introduction to some of the relevant biology and materials science involved in the technical development and current and future use of mNP hyperthermia as clinical cancer therapy.

Photobleaching-based Dosimetry Predicts Deposited Dose in ALA-PpIX PDT of Rodent Esophagus

An improved method to estimate dose to esophageal tissue was investigated in the setting of photodynamic therapy with aminolevulinic acid‐induced protoporphyrin IX (PpIX) treatment. A model of treatment‐induced edema in the esophagus mucosa proved to be a well controlled and useful way to test the dosimetry model, and the light from the treatment laser together with the PpIX fluorescence intensity could be quantified reliably in real time. Dosimetry calculations based upon the detected fluorescence and bleaching kinetics were used to calculate the “effective” dose to the tissue, and a correlation was shown to exist between this metric and the edema induced in the esophagus. The difference between animals with no detectable treatment effect and those with significant edema was predictable based upon the dose calculation. The underlying assumption in the interpretation of the data is that rapid photobleaching of PpIX occurs when there is ample oxygen supply, and this bleaching is not present when oxygen is limited. This leads to the prediction that integration of the light and drug dose, in intervals where appreciable photobleaching occurs, should provide a prediction of the relative dose of singlet oxygen produced. This detection system and rodent model can be used for prospective dosimetry studies that focus on optimization of esophageal PDT.

In vivo modeling of interstitial pressure in the brain under surgical load using finite elements

Current brain deformation models have predominantly reflected solid constitutive relationships generated from empirical ex vivo data and have largely overlooked interstitial hydrodynamic effects. In the context of a technique to update images intraoperatively for image-guided neuronavigation, we have developed and quantified the deformation characteristics of a three-dimensional porous media finite element model of brain deformation in vivo. Results have demonstrated at least 75-85 percent predictive capability, but have also indicated that interstitial hydrodynamics are important. In this paper we investigate interstitial pressure transient behavior in brain tissue when subjected to an acute surgical load consistent with neurosurgical events. Data are presented from three in vivo porcine experiments where subsurface tissue deformation and interhemispheric pressure gradients were measured under conditions of an applied mechanical deformation and then compared to calculations with our three-dimensional brain model. Results demonstrate that porous-media consolidation captures the hydraulic behavior of brain tissue subjected to comparable surgical loads and that the experimental protocol causes minimal trauma to porcine brain tissue. Working values for hydraulic conductivity of white and gray matter are also reported and an assessment of transient pressure gradient effects with respect to deformation is provided.

Multiepitope HER2 Targeting Enhances Photoimmunotherapy of HER2-Overexpressing Cancer Cells with Pyropheophorbide-a Immunoconjugates

Multi-targeting strategies improve the efficacy of antibody and immunotoxin therapies but have not yet been thoroughly explored for HER2-based cancer treatments. We investigated multi-epitope HER2 targeting to boost photosensitizer immunoconjugate uptake as a way of enhancing photoimmunotherapy. Photoimmunotherapy may allow targeted photodynamic destruction of malignancies and may also potentiate anticancer antibodies. However, one obstacle preventing its clinical use is the delivery of enough photosensitizer immunoconjugates to target cells. Anti-HER2 photosensitizer immunoconjugates were constructed from two monoclonal antibodies (mAb), HER50 and HER66, using a novel method originally developed to label photosensitizer immunoconjugates with the photosensitizer, benzoporphyrin derivative verteporfin. Photosensitizer immunoconjugates were labeled instead with a promising alternative photosensitizer, pyropheophorbide-a (PPa), which required only minor changes to the conjugation procedure. Uptake and phototoxicity experiments using human cancer cells were conducted with the photosensitizer immunoconjugates and, for comparison, with free PPa. SK-BR-3 and SK-OV-3 cells served as HER2-overexpressing target cells. MDA-MB-468 cells served as HER2-nonexpressing control cells. Photosensitizer immunoconjugates with PPa/mAb molar ratios up to approximately 10 specifically targeted and photodynamically killed HER2-overexpressing cells. On a per mole basis, photosensitizer immunoconjugates were less phototoxic than free PPa, but photosensitizer immunoconjugates were selective for target cells whereas free PPa was not. Multiepitope targeted photoimmunotherapy with a HER50 and HER66 photosensitizer immunoconjugate mixture was significantly more effective than single-epitope targeted photoimmunotherapy with a single anti-HER2 photosensitizer immunoconjugate, provided photosensitizer immunoconjugate binding was saturated. This study shows that multiepitope targeting enhances HER2-targeted photoimmunotherapy and maintains a high degree of specificity. Consequently, it seems that multitargeted photoimmunotherapy should also be useful against cancers that overexpress other receptors.

Assessment of Photosensitizer Dosimetry and Tissue Damage Assay for Photodynamic Therapy in Advanced-stage Tumors¶

Photodynamic therapy (PDT) efficacy is a complex function of tissue sensitivity, photosensitizer (PS) uptake, tissue oxygen concentration, delivered light dose and some other parameters. To better understand the mechanisms and optimization of PDT treatment, we assessed two techniques for quantifying tissue PS concentration and two methods for quantifying pathological tumor damage. The two methods used to determine tissue PS concentration kinetic were in vivo fluorescence probe and ex vivo chemical extraction. Both methods show that the highest tumor to normal tissue PS uptake ratio appears 4 h after PS administration. Two different histopathologic techniques were used to quantify tumor and normal tissue damage. A planimetry assessment of regional tumor necrosis demonstrated a linear relationship with increasing light dose. However, in large murine tumors this finding was complicated by the presence of significant spontaneous necrosis. A second method (densitometry) assessed cell death by nuclear size and density. With some exceptions the densitometry method generally supported the planimetry results. Although the densitometry method is potentially more accurate, it has greater potential subjectivity. Finally, our research suggests that the tools or methods we are studying for quantifying PS levels and tissue damage are necessary for the understanding of PDT effect and therapeutic ratio in experimental in vivo tumor research.

Local hyperthermia treatment of tumors induces CD8+ T cell-mediated resistance against distal and secondary tumors

UNLABELLED Combinatorial use of iron oxide nanoparticles (IONPs) and an alternating magnetic field (AMF) can induce local hyperthermia in tumors in a controlled and uniform manner. Heating B16 primary tumors at 43°C for 30 min activated dendritic cells (DCs) and subsequently CD8(+) T cells in the draining lymph node (dLN) and conferred resistance against rechallenge with B16 (but not unrelated Lewis Lung carcinoma) given 7 days post hyperthermia on both the primary tumor side and the contralateral side in a CD8(+) T cell-dependent manner. Mice with heated primary tumors also resisted rechallenge given 30 days post hyperthermia. Mice with larger heated primary tumors had greater resistance to secondary tumors. No rechallenge resistance occurred when tumors were heated at 45°C. Our results demonstrate the promising potential of local hyperthermia treatment applied to identified tumors in inducing anti-tumor immune responses that reduce the risk of recurrence and metastasis. FROM THE CLINICAL EDITOR Local heating of tumors via iron oxide NPs and an alternating magnetic field led to activation of anti-cancer CD8 T cells, which resulted in resistance against re-challenge and greater resistance to secondary tumors. Similar local heating-based strategies may become an important weapon in enhancing tumor elimination via a naturally existing but attenuated immune response.