Ultrasound-Stimulated Microbubble Radiation Enhancement of Tumors: Single-Dose and Fractionated Treatment Evaluation

The use of ultrasound-stimulated microbubble therapy has successfully been used to target tumor vasculature and enhance the effects of radiation therapy in tumor xenografts in mice. Here, we further investigate this treatment using larger, more clinically relevant tumor mod- els. New Zealand white rabbits bearing prostate tumor (PC3) xenografts received a single treatment of either ultrasound-stimulated microbubbles (USMB), ionizing radiation (XRT; 8Gy), or a combination of both treatments (USMB+XRT). Treatment outcome was evalu- ated 24 hours after treatment using histopathology, immunolabeling, 3D Doppler ultrasound and photoacoustic imaging. A second cohort of rabbits received multiple treatments over a period of three weeks, where USMB treatments were delivered twice weekly with daily XRT treatments to deliver a fractionated 2Gy dose five days per week. A significant decrease in vascular function, observed through immunolabeling of vascular endothelial cells, was observed in tumors receiving the combined treatment (USMB+XRT) compared to control and single treatment groups. This was associated with an increase in cell death as observed through in situ end labeling (ISEL), a decrease in vascular index measured by Power Dopp- ler imaging, and a decrease in oxygen saturation. In rabbits undergoing the long-term fractionated combined treatment, a significant growth delay was observed after 1 week and a significant reduction in tumor size was observed after 3 weeks with combined therapy. Results demonstrated an enhancement of radiation effect and superior anti-tumor effect of the combination of USMB+XRT compared to the single treatments alone. Tumor growth was maximally inhibited with fractionated radiotherapy combined with the ultrasound-stimulated microbubble-based therapy

Quantitative ultrasound imaging of therapy response in bladder cancer in vivo.

Background and aimsQuantitative ultrasound (QUS) was investigated to monitor bladder cancer treatment response in vivo and to evaluate tumor cell death from combined treatments using ultrasound-stimulated microbubbles and radiation therapy.MethodsTumor-bearing mice (n=45), with bladder cancer xenografts (HT- 1376) were exposed to 9 treatment conditions consisting of variable concentrations of ultrasound-stimulated Definity microbubbles [nil, low (1%), high (3%)], combined with single fractionated doses of radiation (0 Gy, 2 Gy, 8 Gy). High frequency (25 MHz) ultrasound was used to collect the raw radiofrequency (RF) data of the backscatter signal from tumors prior to, and 24 hours after treatment in order to obtain QUS parameters. The calculated QUS spectral parameters included the mid-band fit (MBF), and 0-MHz intercept (SI) using a linear regression analysis of the normalized power spectrum.Results and conclusionsThere were maximal increases in QUS parameters following treatments with high concentration microbubbles combined with 8 Gy radiation: (ΔMBF = +6.41 ± 1.40 (±SD) dBr and SI= + 7.01 ± 1.20 (±SD) dBr. Histological data revealed increased cell death, and a reduction in nuclear size with treatments, which was mirrored by changes in quantitative ultrasound parameters. QUS demonstrated markers to detect treatment effects in bladder tumors in vivo

Ontogenetic and phylogenetic development of the endocrine pancreas (islet organ) in fishes

The morphology of the gastroenteropancreatic (GEP) system of fishes was reviewed with the objective of providing the phylogenetic and ontogenetic development of the system in this vertebrate group, which includes agnathans and gnathostome cartilaginous, actinoptyerygian, and sarcopterygian fishes. Particular emphasis is placed on the fish homolog of the endocrine pancreas of other vertebrates, which is referred to as the islet organ. The one-hormone islet organ (B cells) of larval lampreys is the most basic pattern seen among a free-living vertebrate, with the two-hormone islet organ (B and D cells) of hagfish and the three-hormone islet organ (B, D, and F cells) of adult lampreys implying a phylogenetic trend toward the classic four-hormone islet tissue (B, D, F, and A cells) in most other fishes. An earlier stage in the development of this phylogenetic sequence in vertebrates may have been the restriction of islet-type hormones to the alimentary canal, like that seen in protochordates. The relationship of the islet organ to exocrine pancreatic tissue, or its equivalent, is variable among bony, cartilaginous, and agnathan fishes and is likely a manifestation of the early divergence of these piscine groups. Variations in pancreatic morphology between individuals of subgroups within both the lamprey and chondrichthyan taxa are consistent with their evolutionary distance. A comparison of the distribution and degree of concentration of the components of the islet organ among teleosts indicates a diffuse distribution of relatively small islets in the generalized euteleosts and the tendency for the concentration into Brockmann bodies of large (principal) islets (with or without secondary islets) in the more derived forms. The holostean actinopterygians (Amiiformes and Semiontiformes) share with the basal teleosts (osteoglossomorphs, elopomorphs) the diffuse arrangement of the components of the islet organ that is seen in generalized euteleosts. Since principal islets are also present in adult lampreys the question arises whether principal islets are a derived or a generalized feature among teleosts. There is a paucity of studies on the ontogeny of the GEP system in fish but it has been noted that the timing of the appearance of the islet cell types parallels the time that they appear during phylogeny; the theory of recapitulation has been revisited. It is stressed that the lamprey life cycle provides a good opportunity for studying the development of the GEP system. There are now several markers of cell differentiation in the mammalian endocrine pancreas which would be useful for investigating the development of the islet organ and cells of the remaining GEP system in fish

Ultrastructure and immunocytochemistry of the islet organ of Osteoglossomorpha (Teleostei)

Both routine electron microscopy and immunocytochemistry with protein A–gold were used to identify the cell types within the islet organs of four species of teleosts (Osteoglossum bicirrhosum, Pantodon buchholzi, Notopterus chitala, and Gnathonemus petersii) within Osteoglossomorpha, a subdivision with an ancient lineage. Four primary endocrine cell types, A, B, D, and F, were identified within the islets of the four species examined. The B- and D-cells were located mainly in the central core of the islet in the four species. In general, the A-cells were located at the islet periphery in all of the four species but in P. buchholzi and N. chitala they were also differently distributed toward the islet core. F-cells were present only at the islet periphery. Granules of B-cells in three species had a relatively homogeneous shape of the matrix core, but in O. bicirrhosum, the shape varied greatly. Variation in matrix shape of B-cell granules may indicate a different conformation of insulin molecules among at least some species of osteoglossomorphs, and this observation may have some taxonomic significance. Two somatostatin-containing (SST) D-cell types (D1 and DX) with granules of different shape were observed in all four species of osteoglossomorphs. The granules of the two D-cells immunostained either with anti-SST-25 and anti-SST-14 (D1-cells) or with anti-SST-34 (DX-cells). Immunocytochemistry confirmed that A-cells, containing glucagon-family peptides, and F-cells, containing peptides of the pancreatic polypeptide family, had granules of different shape. The cells of the islet organs of these osteoglossomorphs are more similar to those in more derived teleosts than they are to those of nonteleost actinopterygians

Characterization of the gastro-entero-pancreatic system of osteoglossomorpha, an immunohistochemical, immunocytochemical, and molecular study

grantor: University of TorontoThe principal objectives of this study were twofold: first, to compare the distribution and structure of the GEP system of the osteoglossomorphs with that of the other teleosts and with vertebrates in general; second, to examine the possibility of using the primary structure of the GEP system peptides in a phylogenetic analysis. To fulfill these objectives several approaches were used, including histology, immunohisto- and immunocytochemistry, and molecular cloning. The results indicated that an islet organ (endocrine pancreas) with diffusely scattered islets consists of four cell types (A, B, D and F); there are two D-cell subtypes. Endocrine cells which produce some of the same peptides as the islet cells were also observed within the intestinal epithelia; some variation occurs among the species. For example, insulin was detected in cells within the intestinal and stomach epithelia of 'Pantodon bucholzi ', and infrequently in the intestinal epithelia of 'Osteoglossum bicirrhosum' and 'Gnathonemus petersii', but not in the other species. In the molecular biology studies, preproinsulin and the preprosomatostatin sequences were deduced from the corresponding cDNA sequences and were used in the phylogenetic analysis. The phylogenetic analysis of the preproinsulin sequences confirmed the monophyletic grouping of the osteoglossomorphs, and also indicated that the osteoglossomorphs are not the most basal living teleosts as previously thought, but may be more generalized teleosts. In addition, the phylogenetic analysis showed the elephantnose to be closely related to the feather fin knife fish, and the arawana to be closely related to the butterfly fish. These data are consistent with previous reports (Lauder and Liem 1983; Li and Wilson 1996). The intragroup relationship was confirmed by phylogenetic analysis using sequences of cDNA of preprosomatostatin. These results indicated that the primary structure of the GEP system peptides is useful for inferring phylogenetic analysis to some extent, but also that the sequence analysis of more than one peptide should be considered in such analyses. This study suggests that there is a generalized position of the GEP system in comparison with that of the other teleosts.Ph.D

Molecular cloning of preproinsulin cDNAs from several osteoglossomorphs and a cyprinid

Several preproinsulin cDNAs were isolated and characterized from four members of the Osteoglossomorpha (an ancient teleost group); Osteoglossum bicirrhosum (arawana), Pantodon buchholzi (butterfly fish), Notopterus chitala (feather fin knife fish), Hiodon alosoides (goldeye) and Gnathonemus petersii (elephantnose). In addition, we isolated and characterized the preproinsulin cDNA from Catostomus commersoni (white sucker, as a representative of a generalized teleost). The comparative analysis of the sequences revealed conservation of the cystine residues known to be involved in the formation of the disulfide bridges, as well as residues involved in the hexamer formation, except for B-17 in the butterfly fish, the arawana and the goldeye. However, the N-terminus of the B-chain was very weakly conserved among the species studied. Residues known to be significant for maintaining receptor-binding conformation and those known to comprise the receptor-binding domain were all conserved, except for a conservative substitution at B13, aspartate substituted glutamate in the arawana, goldeye, butterfly fish and white sucker, and at B16, phenylalanine substituted tyrosine in the elephantnose. Phylogenetic analysis of the sequences revealed a monophyletic grouping of the osteoglossomorphs, and showed that they were not the most basal living teleost. Comparative sequence analysis of preproinsulins among the osteoglossomorphs was useful in assessment of intergroup relationship, relating elephantnose with the feather fin knife fish and the arawana, butterfly fish, and goldeye. This arrangement of species is consistent with relationships based on other more classical parameters, except for the goldeye which was assessed as being sister to all the osteoglossomorphs. The white sucker was grouped with the common carp and both are cyprinids

The endocrine cells in the gastroenteropancreatic system of the bowfin, amia calva l.: an immunohistochemical, ultrastructural, and immunocytochemical analysis

The gastroenteropancreatic (GEP) endocrine system of bowfin (Amia calva) was described using light and electron microscopy and immunological methods. The islet organ (endocrine pancreas) consists of diffusely scattered, mostly small islets and isolated patches of cells among and within the exocrine acini. The islets are composed of abundant, centrally located B cells immunoreactive to bovine and lamprey insulin antisera and D cells showing a widespread distribution and specificity to somatostatin antibodies. A and F cells are present at the very periphery of the islets and are immunoreactive with antisera against glucagon (and glucagon-like peptide) and several peptides of the pancreatic polypeptide (PP)-family, respectively. The peptides of the two families usually collocates within the same peripheral islet cells and are the most common immunoreactive peptides present in the extra-islet tissue. Immunocytochemistry and fine structural observations characterised the granule morphology for B and D cells and identified two cell types with granules immunoreactive to glucagon antisera. These two putative A cells had similar granules, which were distinct from either B or D cells, but one of the cells had rod-shaped cytoplasmic inclusions within cisternae of what appeared to be rough endoplasmic reticulum. The inclusions were not immunoreactive to either insulin or glucagon antisera. Only small numbers of cells in the stomach and intestine immunoreacted to antisera against somatostatin, glucagon, and PP-family peptides. The paucity of these cells was reflected in the low concentrations of these peptides in intestinal extracts. The GEP system of bowfin is not unlike that of other actinopterygian fishes, but there are some marked differences that may reflect the antiquity of this system and/or may be a consequence of the ontogeny of this system in this species

Effects of insulin on lipid metabolism of larvae and metamorphosing landlocked sea lamprey,Petromyzon marinus

This study was designed to examine the role of insulin (INS) in regulating changes in lipid metabolism of larval and metamorphosing landlocked lamprey,Petromyzon marinus.Larvae and stage 6 metamorphosing individuals were injected intraperitoneally once per day for 2 days with either saline (0.6%), bovine INS (100 ng/g body weight), or alloxan (0.2 mg/g body weight). Insulin administration resulted in depressed plasma fatty acid (FA) levels, whereas alloxan injection elevated plasma FA levels at both life cycle intervals. In larvae, INS-induced hypolipidemia was attended by increased lipid concentration in kidney and muscle, reduced rates of lipolysis in kidney, liver, and muscle (as indicated by decreased triacylglycerol lipase activity), and, to a lesser extent, by higher rates of lipogenesis in kidney and muscle (as evidenced by higher acetyl-CoA carboxylase and/or diacylglycerol acyltransferase activities). In general, the effects of alloxan were opposite of those of INS. The alloxan-induced increase in plasma FA was supported by an enhanced rate of lipolysis in the kidney, a relatively lower rate of fatty acid synthesis in kidney, liver, and muscle, and a relatively lower renal rate of TG synthesis. In stage 6 metamorphosing lamprey, the INS-induced decline in plasma FA was attended by reduced renal and hepatic rates of lipolysis and by enhanced lipogenesis, as indicated by increased renal and hepatic rates ofde novofatty acid synthesis and hepatic and muscular rates of TG synthesis. In contrast, the increase in plasma FA induced by alloxan in stage 6 animals was supported by reduced TG synthsis in liver. Immunocytochemistry revealed that alloxan was not cytotoxic to pancreatic β cells, suggesting that the effects of alloxan were extrapancreatic in the time frame of our study. Because insulin-induced lipogenesis and antilipolysis is similar to the pattern of lipid metabolism (phase I) displayed by lamprey during their spontaneous metamorphosis, INS may play a role, possibly in concert with other factors, in coordinating metamorphosis-associated changes in lipid metabolism

Cellular characterization of ultrasound-stimulated microbubble radiation enhancement in a prostate cancer xenograft model

Tumor radiation resistance poses a major obstacle in achieving an optimal outcome in radiation therapy. In the current study, we characterize a novel therapeutic approach that combines ultrasound-driven microbubbles with radiation to increase treatment responses in a prostate cancer xenograft model in mice. Tumor response to ultrasound-driven microbubbles and radiation was assessed 24 hours after treatment, which consisted of radiation treatments alone (2 Gy or 8 Gy) or ultrasound-stimulated microbubbles only, or a combination of radiation and ultrasound-stimulated microbubbles. Immunohistochemical analysis using in situ end labeling (ISEL) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) revealed increased cell death within tumors exposed to combined treatments compared with untreated tumors or tumors exposed to radiation alone. Several biomarkers were investigated to evaluate cell proliferation (Ki67), blood leakage (factor VIII), angiogenesis (cluster of differentiation molecule CD31), ceramide-formation, angiogenesis signaling [vascular endothelial growth factor (VEGF)], oxygen limitation (prolyl hydroxylase PHD2) and DNA damage/repair (γH2AX). Results demonstrated reduced vascularity due to vascular disruption by ultrasound-stimulated microbubbles, increased ceramide production and increased DNA damage of tumor cells, despite decreased tumor oxygenation with significantly less proliferating cells in the combined treatments. This combined approach could be a feasible option as a novel enhancing approach in radiation therapy