The effect of flow on blood oxygen level dependent (R*2 ) MRI of orthotopic lung tumors.

PURPOSE: Blood oxygen level dependent (BOLD) MRI based on R2* measurements can provide insights into tumor vascular oxygenation. However, measurements are susceptible to blood flow, which may vary accompanying a hyperoxic gas challenge. We investigated flow sensitivity by comparing R2* measurements with and without flow suppression (fs) in 2 orthotopic lung xenograft tumor models. METHODS: H460 (n = 20) and A549 (n = 20) human lung tumor xenografts were induced by surgical implantation of cancer cells in the right lung of nude rats. MRI was performed at 4.7T after tumors reached 5 to 8 mm in diameter. A multiecho gradient echo MRI sequence was acquired with and without spatial saturation bands on each side of the imaging plane to evaluate the effect of flow on R2* . fs and non-fs R2* MRI measurements were interleaved during an oxygen breathing challenge (from air to 100% O2 ). T2* -weighted signal intensity changes (ΔSI(%)) and R2* measurements were obtained for regions of interest and on a voxel-by-voxel basis and discrepancies quantified with Bland-Altman analysis. RESULTS: Flow suppression affected ΔSI(%) and R2* measurements in each tumor model. Average discrepancy and limits of agreement from Bland-Altman analyses revealed greater flow-related bias in A549 than H460. CONCLUSION: The effect of flow on R2* , and hence BOLD, was tumor model dependent with measurements being more sensitive in well-perfused A549 tumors.

Antibody targeting of phosphatidylserine for the detection and immunotherapy of cancer.

Phosphatidylserine (PS) is a negatively charged phospholipid in all eukaryotic cells that is actively sequestered to the inner leaflet of the cell membrane. Exposure of PS on apoptotic cells is a normal physiological process that triggers their rapid removal by phagocytic engulfment under noninflammatory conditions via receptors primarily expressed on immune cells. PS is aberrantly exposed in the tumor microenvironment and contributes to the overall immunosuppressive signals that antagonize the development of local and systemic antitumor immune responses. PS-mediated immunosuppression in the tumor microenvironment is further exacerbated by chemotherapy and radiation treatments that result in increased levels of PS on dying cells and necrotic tissue. Antibodies targeting PS localize to tumors and block PS-mediated immunosuppression. Targeting exposed PS in the tumor microenvironment may be a novel approach to enhance immune responses to cancer.

Effective Rat Lung Tumor Model for Stereotactic Body Radiation Therapy.

Stereotactic body radiation therapy (SBRT) has found an important role in the treatment of patients with non-small cell lung cancer, demonstrating improvements in dose distribution and even tumor cure rates, particularly for early-stage disease. Despite its emerging clinical efficacy, SBRT has primarily evolved due to advances in medical imaging and more accurate dose delivery, leaving a void in knowledge of the fundamental biological mechanisms underlying its activity. Thus, there is a critical need for the development of orthotropic animal models to further probe the biology associated with high-dose-per-fraction treatment typical of SBRT. We report here on an improved surgically based methodology for generating solitary intrapulmonary nodule tumors, which can be treated with simulated SBRT using the X-RAD 225Cx small animal irradiator and Small Animal RadioTherapy (SmART) Plan treatment system. Over 90% of rats developed solitary tumors in the right lung. Furthermore, the tumor response to radiation was monitored noninvasively via bioluminescence imaging (BLI), and complete ablation of tumor growth was achieved with 36 Gy (3 fractions of 12 Gy each). We report a reproducible, orthotopic, clinically relevant lung tumor model, which better mimics patient treatment regimens. This system can be utilized to further explore the underlying biological mechanisms relevant to SBRT and high-dose-per-fraction radiation exposure and to provide a useful model to explore the efficacy of radiation modifiers in the treatment of non-small cell lung cancer.

Uni-directional ciliary membrane protein trafficking by a cytoplasmic retrograde IFT motor and ciliary ectosome shedding.

The role of the primary cilium in key signaling pathways depends on dynamic regulation of ciliary membrane protein composition, yet we know little about the motors or membrane events that regulate ciliary membrane protein trafficking in existing organelles. Recently, we showed that cilium-generated signaling in Chlamydomonas induced rapid, anterograde IFT-independent, cytoplasmic microtubule-dependent redistribution of the membrane polypeptide, SAG1-C65, from the plasma membrane to the periciliary region and the ciliary membrane. Here, we report that the retrograde IFT motor, cytoplasmic dynein 1b, is required in the cytoplasm for this rapid redistribution. Furthermore, signaling-induced trafficking of SAG1-C65 into cilia is unidirectional and the entire complement of cellular SAG1-C65 is shed during signaling and can be recovered in the form of ciliary ectosomes that retain signal-inducing activity. Thus, during signaling, cells regulate ciliary membrane protein composition through cytoplasmic action of the retrograde IFT motor and shedding of ciliary ectosomes.

Serotonergic modulation of respiratory motor output during tadpole development.

To test the hypothesis that serotonin (5-hydroxytryptamine; 5-HT)-receptor activation elicits age-dependent changes in respiratory motor output, we compared the effects of 5-HT bath application (5-HT concentration = 0.5-25 microM) onto in vitro brain stem preparations from pre- and postmetamorphic bullfrog tadpoles. Recording of motor output related to gill and lung ventilation showed that 5-HT elicits a dose-dependent depression of gill burst frequency in both groups. In contrast, the lung burst frequency response was stage dependent; an increase in lung burst frequency at low 5-HT concentration (< or =0.5 microM) was observed only in the postmetamorphic group. Higher 5-HT concentrations decreased lung burst frequency in all preparations. Gill burst frequency attenuation is mediated (at least in part) by 5-HT(1A)-receptor activation in an age-dependent fashion. We conclude that serotonergic modulation of respiratory motor output 1) changes during tadpole development and 2) is distinct for gill and lung ventilation.

Role of 5-HT2A/C receptors in serotonergic modulation of respiratory motor output during tadpole development.

We tested the hypothesis that 5-HT(2A/C) receptors contribute to the modulation of respiratory motor output elicited by serotonin bath-application in the in vitro brainstem preparation from bullfrog tadpoles. Preparations were superfused with (1) a selective 5-HT(2A/C) receptor agonist (DOI, 0.5-25 microM) or (2) 5-HT in presence of a non-selective 5-HT(2A/C) receptor antagonist (methysergide maleate, 10 microM). Stage-related differences in 5-HT(2A/C) modulation were assessed by performing experiments on two groups: pre- and post-metamorphic tadpoles. 5-HT(2A/C) receptor activation with DOI did not alter either gill or lung burst frequency. Methysergide did not prevent the gill burst frequency attenuation elicited by 5-HT. Our results show no apparent contribution of the 5-HT(2A/C) receptor subtypes to the serotonergic modulation of either gill- or lung-related neural activity in either group. While 5-HT(2A/C) receptors are involved in the modulation of respiratory motor output in mammals, these receptor subtypes do not appear to play a significant role in this species.

[Amphibians as a model system for the investigation of respiratory control development]. / Un modèle amphibien pour l'étude du développement du contrôle de la respiration.

Recent medical advances have made it possible for babies to survive premature birth at increasingly earlier developmental stages. This population requires costly and sophisticated medical care to address the problems associated with immaturity of the respiratory system. In addition to pulmonary complications, respiratory instability and apnea reflecting immaturity of the respiratory control system are major causes of hospitalization and morbidity in this highly vulnerable population. These medical concerns, combined with the curiosity of physiologists, have contributed to the expansion of research in respiratory neurobiology. While most researchers working in this field commonly use rodents as an animal model, recent research using in vitro brainstem preparation from bullfrogs (Rana catesbeiana) have revealed the technical advantages of this animal model, and shown that the basic principles underlying respiratory control and its ontogeny are very similar between these two groups of vertebrates. The present review highlights the recent advances in the area of research with a focus on intermittent (episodic) breathing and the role of serotonergic and GABAergic modulation of respiratory activity during development.

Phosphatidylserine-targeted bimodal liposomal nanoparticles for in vivo imaging of breast cancer in mice.

Phosphatidylserine (PS) that is normally constrained to the inner plasma membrane becomes exposed on the surface of endothelial cells (ECs) in tumor vasculature. In the present study, we report the development of a novel tumor vasculature-targeted liposomal nanoprobe by conjugating a human monoclonal antibody, PGN635 that specifically targets PS to polyethylene glycol-coated liposomes. MR contrast, superparamagnetic iron oxide nanoparticles (SPIO) were packed into the core of liposomes, while near-infrared dye, DiR was incorporated into the lipophilic bilayer. The liposomal nanoprobe PGN-L-IO/DiR was fully characterized, and its binding specificity and subsequent internalization into PS-exposed vascular ECs was confirmed by in vitro MRI and histological staining. In vivo longitudinal MRI and optical imaging were performed after i.v. injection of the liposomal nanoprobes into mice bearing breast MDA-MB231 tumors. At 9.4T, T2-weighted MRI detected drastic reduction on signal intensity and T2 values of tumors at 24h. Ionizing radiation significantly increased PS exposure on tumor vascular ECs, resulting in a further MRI signal loss of tumors. Concurrent with MRI, optical imaging revealed a clear tumor contrast at 24h. Intriguingly, PGN-L-IO/DiR exhibited distinct pharmacokinetics and biodistribution with significantly reduced accumulations in liver or spleen. Localization of PGN-L-IO/DiR to tumor was antigen specific, since a control probe of irrelevant specificity showed minimal accumulation in the tumors. Our studies indicate that PS-targeted liposomes may provide a useful platform for tumor-targeted delivery of imaging contrast agents or potentially anti-cancer drugs for cancer theranostics.

Regulated membrane protein entry into flagella is facilitated by cytoplasmic microtubules and does not require IFT.

The membrane protein composition of the primary cilium, a key sensory organelle, is dynamically regulated during cilium-generated signaling [1, 2]. During ciliogenesis, ciliary membrane proteins, along with structural and signaling proteins, are carried through the multicomponent, intensely studied ciliary diffusion barrier at the base of the organelle [3-8] by intraflagellar transport (IFT) [9-18]. A favored model is that signaling-triggered accumulation of previously excluded membrane proteins in fully formed cilia [19-21] also requires IFT, but direct evidence is lacking. Here, in studies of regulated entry of a membrane protein into the flagellum of Chlamydomonas, we show that cells use an IFT-independent mechanism to breach the diffusion barrier at the flagellar base. In resting cells, a flagellar signaling component [22], the integral membrane polypeptide SAG1-C65, is uniformly distributed over the plasma membrane and excluded from the flagellar membrane. Flagellar adhesion-induced signaling triggers rapid, striking redistribution of the protein to the apical ends of the cells concomitantly with entry into the flagella. Protein polarization and flagellar enrichment are facilitated by cytoplasmic microtubules. Using a conditional anterograde IFT mutant, we demonstrate that the IFT machinery is not required for regulated SAG1-C65 entry into flagella. Thus, integral membrane proteins can negotiate passage through the ciliary diffusion barrier without the need for a motor.

Effects of medullary Raphé stimulation on fictive lung ventilation during development in Rana catesbeiana.

To better understand serotonergic modulation of air breathing during bullfrog development, we measured changes in fictive lung ventilation frequency associated with focal stimulation of the rostral region of the medullary Raphé neurons. Electrical (3 to 33 Hz) and chemical (glutamate microinjections; 0.5 mol l(-1), 0.3-10 nl) activation of Raphé neurons was performed in brainstem preparations from three developmental stages (pre- and metamorphic tadpoles and adult frogs). Fictive lung ventilation was recorded extracelluarly from the Vth and Xth cranial nerves. Electrical stimulation of Raphé neurons caused a frequency-dependent increase in lung burst frequency in pre-metamorphic tadpoles only. In metamorphic tadpoles, an increase in fictive lung ventilation was observed at 20 Hz only. Electrical stimulation had no effect in preparations from adult frogs. Glutamate microinjections elicited similar responses as a lung burst frequency increase was observed in the pre-metamorphic group only. Regardless of the stimulation technique used, the increase in fictive lung ventilation was attenuated by the selective 5-HT3 antagonist tropisetron (5-20 micromol l(-1)). Results from immunohistochemical analysis of the Raphé region stimulated do not correlate with functional data as the number of 5-HT immunoreactive neurons within this region increases during development. We conclude that, in this preparation, stimulation of lung ventilation by the medullary Raphé is restricted to early (pre-metamorphic) stages.