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The Revolution of Sigmar

The One Where Jewel is a Bad-Ass

Plot Synopsis #16

“I’m tough, I’m ambitious, and I know exactly what I want. If that makes me a bitch, okay.”

16-1: Aftermath

“Is everyone okay?” Brevan asked, rushing over to his allies. After felling the monstrous trees, his companions slowly began to recuperate from their near fatal encounter with a pair of beings that Shuriel had referred to as, “Hangman Trees.” Their noose-like limbs fell limp at the ground, but the mighty plants hadn’t died without leaving their mark on the far from helpless travelers. Zerrik’s once bright and well-polished armor was dulled from exposure to the plant’s digestive track and Shuriel had several dark bruises along his neck where the tree had attempted to hang him. Omar was furiously cleaning the plants’ internal fluids from his blunderbuss’s barrel, and Bippy was nursing her right hand, which had nearly snapped off of her wrist from the force of the tree’s pull. Only Brevan and Jewel had managed to avoid harm, and Jewel was in bright spirits as she swung from atop the tree to the ground, having battled the monster and won without suffering a single scratch.

“I’m fine,” Jewel remarked. “That tree didn’t touch me once!”

“Yeah, well good for you,” Omar grumbled, remembering how not seconds before he was nearly swallowed alive into the bowels of the monstrous tree. “I’m starting to agree with Zerrik. This entire job seems like it’s more trouble than its worth.”

“Perhaps,” Bippy replied sternly as she called upon the forces of life, drenching all present in waves of life energy, healing their wounds. She flexed her wrist and rubbed it one last time, content that it would retain its usefulness. “But if you seek the aid of the Bellytuff clan, you will perform this task for us.”

“Getting the Bellytuffs to move back with the Gullykins would be a huge boost to the town’s population,” Brevan reminded Omar. “They seem to have fared much better then we did, under Jamtoes and their skills at animal herding combined with their hardened combat skills would make them invaluable to the success of the village.”

“This is also true,” Bippy nodded. Brevan would have bet gold that her eyes would be sparkling with pride at his praise if she had any.

“Yes, but do not forget our bargin, Kindlebrin,” Zerrik replied sternly.

“I know, I know. 24 hours. We have plenty of time; we’ve been wandering around for what? Several hours? It’s ten o’lock at the earliest. We have plenty of time and by my mayoral honor, I’ll honor our agreement Zerrik.”

“I do not mean to interupt,” Shuriel interjected courtly. “But I’ve been analyzing the remains of the hangman trees, and their bowels’ occupants would appear that at some point they had managed to engulf some other powerful individuals.”

“You need to engulf an every man dictionary sometime,” Omar grunted as the last of the plant’s stomach acid was cleaned from his weapon. “What the heck you trying to say?”’

“Big tree,” Shuriel responded slowly, emphasizing every sound he spoke so that the slowest ogre could follow his words. “Have goodies in tummy.”

“The spoils of conquest?” Zerrik shouted loudly, the solemnness of his god’s mission forgotten amongst daydreams of gold and fantastic treasures if only for a moment. “Let’s see what treasures await us in the bellies of these beasts!”

“Whoa there, just a second Zerrik,” Shurriel quickly replied, halting the burly man from jumping back into the tree’s stomach. “Let me sprinkle some of my special acid dostabilizer in there. It’ll cancel out the acid so we can poke around in there without melting.” Shuriel rummaged through his pouch for some powders and began throwing handfuls of the stuff inside of the tree, the acid immediately reacting and changing from a bright green to a shade of deep blue.

“Ass of an elf,” Omar muttered as he put his gun and satchel on the ground. Alchemical contraption or not, there was no way he would clean his blunderbuss of stomach fluid again so soon. After several minutes of stomach diving, the party had a handful of magical equipment in front of them; Brevan and Shuriel immediately got to work attempting to identify their properties. In the end, they had found a ring with a potent enchantment that warded the wearer from attacks, a magical glove that allowed the user to store a moderately sized object within it until called upon, a torn and frayed headband that would enhance the wearer’s force of personality, a magical dagger with the ability to spew forth venom on command, and a ring that could be used to counter enemy spellcasting. After some debate and bartering, each member of the group selected an item to their preferences. Omar quickly placed his chosen item, the headband of alluring charisma, on his brow.

“What do you think? Is my personality any more forceful?” Omar snickered, the golden headband twinkling in the faint sunlight.

“Probably not, considering that headband’s probably been in the beast’s stomach for so long that the magic has all but faded from the headband,” Shuriel snickered as he experimented with his choice, the glove of storing. Omar again cursed the alchemist, choosing to keep the headband on his forehead simply to keep his temple covered.

16-2: Visitors from Afar

Once they had sorted through their treasure, the group prepared to set out once more in search of the tainted beings. Looking down at his wrist, Brevan let out a small gasp as he watched the sign that Bippy had placed on his wrist fade slightly.

“I think those things were somehow tied to the forest’s corruption,” Brevan said while still staring at his wrist.

“Well, those are naturally occurring monsters,” Shuriel mused, “But it’s possible that they were awakened by this taint you keep speaking of. Hangman trees typically don’t tolerate each other’s company.”

“The sign is saying to continue straight through,” Brevan replied, nodding slightly at Shuriel’s deductions.

“I will take to the trees once more,” Jewel said calmly, climbing back into the sky among the branches of the forest canopy. Her new, magical dagger swung from side to side as she climbed. “My new weapon needs a name,” she thought to herself as she allowed herself to place more and more distance between herself and her comrades. Lost in her thoughts, she barely noticed when, nearly 100 yards in the distance, a faint, otherworldly light shone through the forests; she guessed that such a light would need a powerful source to shine so brightly in the afternoon. Jewel signaled back to her friends before continuing onward.Shuriel watched Jewel’s movements carefully and quickly relayed to the party what she had told him.

“Jewel is signalling that there’s something ahead. Looks weird. Maybe a bright light or something? Either way, it’s about a 100 yards ahead and she’s going to check it out.”

“This could be exactly what we’re looking for!” Brevan exclaimed as he hastened his steps. Back ahead, Jewel hurried towards the light, curious to see what she would find there. when she finally reached the source of the light, she gasped in horror and disgust.

She was sitting in a tree, high above a clearing. Below, scampering around where small, humanoid creatures with wild hair that muttered and rambled in Undercommon; a language that Jewel was all-too familiar with, having grown up in a drow city located deep within the Underworld. The creatures were called derro; made, deranged little beasts who were all but born with warped minds. Like drow, however, derro were naturally adapted to life in the lightless world below the surface; so why were they here, working in this particular forest clearing? The little wretches wandered back and forth from tree to tree where three large, bulbous sacs of a flesh-like substance was growing. The violet bulbs pulsed with yellow light; the same light Jewel had seen so far away. The light traveled around the bulbs like blood through veins, the bulbs in turn climbing up the trees slowly with every burst of light. There were three bulbs, forming a triangular pattern in the clearing.

“Jewel tried to lean in closer in order to see what exactly the verminous derro were doing to the forest, but her branch creaked ever so slightly strained by her weight. One of the derro; the one closest to her, looked up with madness in its eyes, staring directly at her. It began screaming in Undercommon, “Drow! Drow! I sees the drow1 Kill it, maim it! Let the master dissects it!” Jewel quickly pulled out her loaded crossbow and fired it at the creature, striking it cleanly in the shoulder. It pulled out its own crossbow and launched back its own bolt, piercing Jewel’s armor. She pulled out the bolt quickly and with a measure of pain; the smell told her immediately that it was drenched in spider poison, another favorite the creatures of the Underworld.

“We got ourselves a problem!” Jewel yelled as she reloaded her crossbow. Brevan, Zerrik, Bippy, Shuriel, and Omar ran into the clearing as she called for them, stunned by the alien pods before them. Omar got over the scenery first as he quickly loaded his blunderbuss with a flare and fired it at one of the derro. Though the others weren’t close enough, Omar knew as well as Jewel that the Derro hated light, and the little humanoid began screaming at the top of its lungs about the burning light as it wailed.

“Get some light in here!” Omar yelped. “They’re derro; sunlight burns their flesh right off the bone!”

Zerrik, who had no means to chop apart leaves that were so far away, bellowed as he rushed another derro, striking a clean blow with his ax. Lacking Zerrik’s powerful frame and mighty strength, the blow was a near-fatal one, blood gushing from the little monster’s shoulder wound. It tried to retaliate, but Zerrik’s blow had loosened the creature’s armor straps, causing it to stumble all over itself. The creature momentarily stunned, Bippy tossed a small, golden ring to Zerrik not unlike the one she had given to Brevan earlier. With a nod of understanding, Zerrik slipped the enchanted ring over his metallic gauntlet, the ring adjusting to his body’s armor.

Shuriel rushed into combat alongside Zerrik, branding the fallen angel’s blade that had served him well in the previous encounters. He swung the blade at the derro who had fired its weapon at Jewel, taking a chuck of withered flesh from its shoulder. Brevan, calling upon his magical powers, summoned forth a massive sphere of fire and hurled it up into the treetops at Omar’s cry, bathing the derro in light. Omar was not speaking metaphorically; their flesh literally melted from their bodies as they screamed in pain while being drenched in the poisonous sunlight.

Far from dissuaded, the derro who originally saw Jewel was transfixed on its target, tumbling past Shuriel with surprising ability and ferociously climbing up the tree towards its target. Jewel leaped into the trees and cut apart some additional branches, blasting her pursuer with more sunlight; it screamed but shrugged off the searing rays with determination, carrying a poison-drenched club in its teeth as it climbed ever higher. The flared derro flailed about in agony, jumping from the light that Brevan’s fire had reigned down, charging the closest thing it could see; Bippy. It struck her with its mace, bashing the blind halfling senseless. The other one, seeking vengeance against Zerrik. Blinded by the sunlight, it swung at Zerrick in vain, its feeble blows not leaving a single dent in his armor.

Suddenly, the little derro that had missed Zerrik stared up in the sky and starting wailing with joy at the top of its lungs. The other two quickly followed suit. While Zerrik, Brevan, and Bippy had no idea what the derro were saying, the elves did, and all three looked up into the sky.

“What are they saying Shuriel … oh,” Brevan had asked Shuriel, but his eyes followed Shuriel’s mid-statement. Descending from the sky was a creature unlike anything that any of the six adventures had ever seen; nothing on this world compared to the horrifying sight. Its torso was like a worm’s, its body ending with a tail that swayed back and forth. Six insectoid legs with a set of sharp claws dangling below the creature hung from the torso, and several sets of wings that greatly resembled those of a dragonfly’s hummed louder and louder as the creature descended. Where the creature’s head ought to be, there was only a mass of tendrils that pulsed in rhythm with the bulbs attached to the trees around the clearing. Its head was fungal in appearance as it inflated and deflated in this haunting rhythm.

The creature landed between Shuriel and Zerrik; both could see that it was covered in an array of plate that was inscrbed with runes in an alien language; even hyper-literate Shuriel couldn’t read them. The monster quickly pulled out a syringe and plunged it into Shuriel’s neck; his eyes blinked twice before the elf collapsed and the monster turned its attention to Zerrik, the derro chanting in joy.

“Shuriel’s down!” Zerrik called as he raised his sword against this new challenger. Up in her tree, Jewel gasped, barely noticing that the derro was climbing ever higher into her tree; it took an arced swipe at her with its shortsword that she was only barely able to dodge in time. Bippy swung her mace against the derro that was attacking her, but her life-sensing ‘vision’ was overwhelmed by the presence of the creature and her mace crashed into a large stone, its hilt cracking and splintering from the impact. Upon seeing this new creature, Brevan attempted to magically junx the monstrosity, but its mind was alien and horrible screams filled his eyes as he tried to penetrate the monster’s brain; his spell had no effect. Likewise, Zerrik backed away from the monster and attempted to hold it in place with magic to the same effect; the creature’s mind was too alien for his magic to compel. Omar had his own idea as he pulled out his blunderbuss as blasted the monster with his fun, riping a massive hole in into fungus-like head.

Meanwhile, in the trees Jewel battled with the derro, dueling it high above the ground. The monster snarled at her, spouting insults about her heritage with every thrust and parry of its tiny sword. Jewel parried the attacks expertly with her new dagger and waited for an opening, sinking her rapier into the creature’s chest, then plunging her dagger into its head, killing it immediately. She kicked the corpse from her rapier, allowing it to fall to the ground. From above, she observed the scene below; Shuriel was unconscious and the remaining derro, no longer barred in their actions because her allies were focused on the alien monstrosity, crept towards Shuriel, singing on Undercommon.

“Kick the elf-y, maim the elf-y, slit the elf-y in its throat.
Slice the elf-y, dice the elf-y, wear his pink skin like a coat!”

“Zerrik! They’re going for Shuriel!” Jewel called out in despair. The mighty warrior swung his axe at one of the derro, who was attempting to cut Shuriel’s throat apart, ironically sending the Underworld denizen’s head rolling off of his shoulders. The remaining derro, enraged by its comrade’s death, abandoned Shuriel and tried in vain to beat Zerrik with its club.

Meanwhile, the monster had taken a serve beating between Zerrik’s and Omar’s attacks, coupled with two powerful scorching ray spells; once by Brevan and again by Zerrik, who had the spell store into his blade by Brevan that morning before they left. Despite the merciless beating, the monster looked as strong as ever; especially after it reached into its satchel and pulled out a second syringe, plunging it into itself. Were Shuriel awake, he would have been able to identify the amixture with no problem; Brevan and Zerrick just gaped in awe as the monster’s wounds healed over and it grabbed Shuriel with one of its insectoid legs, seemingly content to fly off with at least one victim and live.

The friends tried to save their companion with little effect; he was too high up for Omar to accurately shoot at it and neither Bippy or Zerrik had no means to attack the creature from afar. Brevan fired a barrage of magic missiles at the monster and although not a single missile missed its mark, their force wasn’t enough to down the recently healed creature. All seemed lost; they would be unable to save Shuriel.

However, there was one thing that Brevan, Omar, and Zerrik had not considered. Though the monster was too high up for any of them to reasonably be able to attack, all its increased height did was bring it ever closer to Jewel. Setting aside her fear and paranoia, she stepped backwards slightly and pushed herself into the air with all of her strength. She went flying and tackled the monster in the sky; grabbing it by its wings. The monster clicked in protest, but Jewel held firm, stabbing her dagger into the monster. The magical dagger radiated green magic and began dripping poison, which immediately took its toll on the monster; its wing nearly severed, it could no longer keep aloft and it plummeted towards the ground, taking Jewel and Shuriel with it.

The monster’s bulbous body worked against it this time; though it took the full blunt impact of the fall, its body cushioned Shuriel from most of the damage and Shuriel danced through the air like a leaf, allowing herself to prevent most of the damage. As she landed, Omar blasted a giant hole into the remaining derro and Zerrik charged up to the fallen monster, writhing weakly in pain, and ended the creature with his blade; cleaving the monster in two.

16-3: Sleeping Shuriel

With the enemies dead and little he could do for his fallen companions, Omar set about looting the corpses of the derro, grabbing one of Shuriel’s bags of holding, picking them clean of all of their possessions, and stuffing them into the bag carelessly.

Meanwhile, Zerrik, Brevan, Bippy, and Jewel were crowded around Shuriel, who was still out cold, despite his fall and resulting injuries. Calling upon the powers of life once more, Bippy healed her companions of their wounds, but no amount of her magical energy could seem to break the spell that had taken hold of Shuriel.

“What exactly did it do to him?” Jewel wondered aloud.

“It pulled out something and stabbed him with it; it looked like it pumped a liquid into his body.” Zerrik replied.

“Do you have any spells that could awaken him?”

“None for today, I’m afraid. We could wait for night to fall so I could restore my powers, but I’m afraid that Brevan and my agreement would end then, and I would leave to find he who my god has deemed must die.” Zerrik replied stoically.

“You mean you’d just as soon abandon him like this?” Jewel lashed out at Zerrik, who took a step back out of reflex. “He would do anything within his power to help you; he flipped when that man in yellow murdered you in cold blood, and you’d sooner abandon him?”

“It isn’t a choice I make lightly,” Zerrik snapped. “But Set has ordained it.”

Meanwhile, Omar had finished gathering the possessions of the dead, so he returned to the group to find Zerrik and Jewel in the middle of their heated discussion. Having only half-brokenheartedly listening to their squabble, he looked down to see Shuriel still unconscious.

“I’m no expert on magic, but couldn’t you just splash some water on him or something?” Jewel and Zerrik looked at each other blankly. Several moments later, after Zerrik had called upon his divine powers to fill a bucket with conjured water, the pair heaved the water across Shuriel’s face. The elf simply slept through the exchange.

“Any other ideas?” Jewel asked coldly.

p, “I could shoot’em with some rock salt. That’ll hurt like hell; maybe it’ll wake him up.” Omar offered.

“Maybe,” Jewel said, stopping to think for a moment. “Maybe if this doesn’t work.” Sowly, Jewel tied her hair behind her hair and crouched atop of Shuriel like a predatory cat. She leaned down close; she could feel the elf’s peaceful breathing; his breath warming her lips. She closed her eyes and hoped for a second that the stories she had heard were true, of magical sleep being broken by a kiss from nobility. Jewel backed away from Shuriel, whose eyes began blinking rapidly. Slowly, his eyes opened and he rubbed them slowly, waking from a deep slumber.

“What … what happened? We killed it? Why am I covered in water?” Shuriel asked as he pushed himself up.

“Damn it, and I wanted to shot him too,” Omar pouted, his brow furrowing as Jewel backed away from Shuriel.

~ End Entry #16

Art by nJoe and

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Friday, March 8, 2013
By Paul Martin

By Darren Weeks
March 8, 2013

Mayoral resolution approved by council gives power to military

Martial Law is defined by Random House dictionary as:

1. the law temporarily imposed upon an area by state or national military forces when civil authority has broken down or during wartime military operations.

2. the law imposed upon a defeated country or occupied territory by the military forces of the occupying power.

In my last article, I explored the Martial Law declaration that was issued in the aftermath of Hurricane Sandy and its devastation. I made the statement that I believe we are beginning to see the unfolding of a new norm. A trend seems to be evolving where local, state, or regional officials are using states of emergency or outright Martial Law declarations to deal with small-scale problems.

Since that time, we have seen three states — Connecticut, Rhode Island and Massachusetts — all issue emergency declarations, restricting travel, due to an anticipated snowstorm. One might ask how could it be that northern states might be so ill-prepared for a major snowstorm so as to warrant the suspension of thousands of individuals’ right to travel? Imagine… snow in the north! In the winter! Who would have thunk it?

Massachusetts Governor Deval Patrick issued an executive order on February 8th, banning all civilian travel with certain very specific exceptions — mainly emergency services, the media, and those contributing to critical infrastructure and resources. Numerous media sources reported that violation of the Deval’s order would carry with it a potential $1,000 fine and up to a year in jail! What if your wife was pregnant and you needed to get her to the hospital? Guess you’re out of luck, huh? What if you have some other emergency? Too bad for you! Your exception to the rule wasn’t mentioned in Dictator Deval’s order.

Aside from the fact that there are countless legitimate reasons for people to be on the roadways during a major snowstorm — many of which couldn’t possibly be foreseen by an executive decree — this “Deval” governor has no business restricting the people’s right to travel. Yet, that is exactly what he did, which is completely unnecessary and should be considered unacceptable to the populace. He should be immediately recalled, and state legislative guidelines should be put into place, making it a criminal offense to use executive orders for purposes such as this. Encouraging people to stay off the roads is fine. Mandating it under threat of massive fines and imprisonment? Not in a free society.

On the heels of that outrage, we now learn that on February 20th, Tucson, Arizona Mayor Jonathan Rothschild — yes, that’s really his name — issued Resolution #22006 (pdf) which declares a state of emergency in the city of Tucson.

Section 1 “recognizes” the leadership of the Davis-Monthan Air Force Base “to make the appropriate decisions when balancing National Security and community needs when it comes to their existing and future military mission and assignments.”

The Rest…HERE

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Saturday, December 11, 2010

Another K-r Review and a "5"s' ISO/IQ Shootout

New K-r Review at DC Review:-

And how the Oly 4/3 E-5 is compared head-to-head to the APS-C K-5:-

Well, can I declare death of the 4/3 system from judging on these? :-o (Although the IQ and colours could be better at the base ISOs.)

Read Also:-

All reported K-r Reviews

All reported K-5 Reviews

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RiceHigh's Pentax Blog by RiceHigh is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License.


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Saturday, June 09, 2007

Ladybookbug: A New Anonymous Book Reviewer

Ladybookbug has joined the ranks of librarians writing reviews of the books she reads. Her blog is titled "On the Bookshelf," and her latest review looks at The Road by Cormac McCarthy. She would not have read the book if she had not heard the author interviewed, but then she was glad that she had.

So far, all but one of her books have been fiction, and most are top sellers. My favorite review, however, was of a first novel, Summer People by Brian Groh. I hope that she tackles more lesser-known authors to help us learn about them.

I added her URL to the Librarian's Book Revoogle, but none of her reviews appear in results yet. It appears that Google does not rank her blog yet. Visit her blog to help it out.

1 comment:

Mark Molckovsky said...
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onsdag 23 november 2011

Barn Find Big Twin Flathead

Back in mid 1970’s Sweden, in the shadows of ABBA’s international breakthrough there was an anonymous gentleman who parked his 1946 Model U in a barn located on the frozen tundra way up north in an ancient village called Vilhelmina, the exact year was 1974. What the anonymous gentleman probably had a hard time predicting was that the Model U would remained parked in the exact same spot for the forthcoming 26 years. So, in November 2010, Agnetha Fältskog still hadn’t committed to a reunion with Björn Ulvaeus, Benny Andersson and Annifrid Lyngstad in order to make ABBA see the light again. But lucky enough my friend Jonas Wickman committed to his passion for the ancient iron and rode all the way up north to make this abandoned 1946 U see the light again. This bike came with the original title and complete documentation, it was sold new in Spånga right outside Stockholm back in 1946, it was bought by a carpenter that used the bike in his job and it’s connected with a 1953 Steib sidecar complete with a carpenter’s toolbox.

The first thing that hit me when I kick started it was that this thing has a lot of compression for a Model U, it felt more like kicking over a stock Panhead. It started right up on first kick and sounded better than any of ABBA’s harmonies and idled lovely, like only a Flathead can. This bike came from the dealer in Spånga, Stockholm with an original Harley sidecar but somewhere in time it got switched out to this beautiful Steib rig. And since Sweden still had left hand traffic back then the sidecar is mounted on the left side. This bike runs strong and it still has the original everything pretty much including paint and tank trim, I would also guess that neither the motor or transmission has ever been taken apart. Nothing beats a Flathead.

5 kommentarer:

  1. yeah man beautiful, thats really cool.

  2. pretty much my dream bike

  3. that is a fantastic find, what a great machine - dig that it's never been dissected...

    ABBA? not so much for this Norte Americano, they make my milk curdle

  4. Nicke you rule man! Got good bike karma...


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Onyx ringtone to your cellphone

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Music Lessons

Our experienced teachers develop a personal connection with students and customize their instruction to help you grow with your needs and interests. We offer instruction for children and adults, from beginners to professionals.
Private lessons are available through individual instructors.

From voice to guitar and violin to drum lessons, it’s never too late to hone your craft or learn a new instrument. Financial assistance is available with documented need. If you have questions about signing up for private music lessons at RME, contact
Ellis Kell – Director of Programming & Community Outreach at 563-326-1333 ext.113 or visit


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Open Access

Vascular responses to radiotherapy and androgen-deprivation therapy in experimental prostate cancer

  • Kathrine Røe1, 2, 3Email author,
  • Lars TG Mikalsen4,
  • Albert J van der Kogel5, 6,
  • Johan Bussink5,
  • Heidi Lyng1,
  • Anne H Ree3, 2,
  • Laure Marignol7 and
  • Dag R Olsen8
Radiation Oncology20127:75

DOI: 10.1186/1748-717X-7-75

Received: 1 February 2012

Accepted: 26 April 2012

Published: 23 May 2012

Radiotherapy (RT) and androgen-deprivation therapy (ADT) are standard treatments for advanced prostate cancer (PC). Tumor vascularization is recognized as an important physiological feature likely to impact on both RT and ADT response, and this study therefore aimed to characterize the vascular responses to RT and ADT in experimental PC.

Using mice implanted with CWR22 PC xenografts, vascular responses to RT and ADT by castration were visualized in vivo by DCE MRI, before contrast-enhancement curves were analyzed both semi-quantitatively and by pharmacokinetic modeling. Extracted image parameters were correlated to the results from ex vivo quantitative fluorescent immunohistochemical analysis (qIHC) of tumor vascularization (9 F1), perfusion (Hoechst 33342), and hypoxia (pimonidazole), performed on tissue sections made from tumors excised directly after DCE MRI.

Compared to untreated (Ctrl) tumors, an improved and highly functional vascularization was detected in androgen-deprived (AD) tumors, reflected by increases in DCE MRI parameters and by increased number of vessels (VN), vessel density ( VD), and vessel area fraction ( VF) from qIHC. Although total hypoxic fractions ( HF) did not change, estimated acute hypoxia scores ( AHS) – the proportion of hypoxia staining within 50 μm from perfusion staining – were increased in AD tumors compared to in Ctrl tumors. Five to six months after ADT renewed castration-resistant (CR) tumor growth appeared with an even further enhanced tumor vascularization. Compared to the large vascular changes induced by ADT, RT induced minor vascular changes. Correlating DCE MRI and qIHC parameters unveiled the semi-quantitative parameters area under curve ( AUC) from initial time-points to strongly correlate with VD and VF, whereas estimation of vessel size ( VS) by DCE MRI required pharmacokinetic modeling. HF was not correlated to any DCE MRI parameter, however, AHS may be estimated after pharmacokinetic modeling. Interestingly, such modeling also detected tumor necrosis very strongly.

DCE MRI reliably allows non-invasive assessment of tumors’ vascular function. The findings of increased tumor vascularization after ADT encourage further studies into whether these changes are beneficial for combined RT, or if treatment with anti-angiogenic therapy may be a strategy to improve the therapeutic efficacy of ADT in advanced PC.

Prostate cancer Androgen-deprivation therapy Radiotherapy Immunohistochemistry Dynamic contrast-enhanced MRI

Androgens play key regulatory functions in prostate cancer (PC) growth and proliferation. Androgen-deprivation therapy (ADT) is established as an integral component of contemporary management of advanced PC, providing reduction of androgen-sensitive tumor cells and symptomatic relief for the patients [1]. However, the lack of durable remission indicates that current ADT strategies are suboptimal, or that successful treatment outcome may require ADT to be combined with other therapeutic interventions. Specifically, the combination of ADT with radiotherapy (RT) has proven efficacious [2, 3], yet the optimal timing of RT in relation to ADT remains unclear.

Tumor vascularization is an important physiological feature of tumor tissue, influencing both RT and ADT response. Adequate tumor oxygenation results in enhanced radical formation following RT, inducing irreversible DNA damage and subsequent tumor cell death [4]. Well-vascularized tumors are more likely to be well oxygenated, in contrast to poorly vascularized tumors often suffering from tumor hypoxia, reducing the efficacy of combined treatments, including RT. Moreover, ADT affects tumor vascularization by its direct influence on androgen-sensitive, endothelial cells. Although the endothelial cells express androgen receptors providing paracrine signaling and vascular support facilitating recurrent growth, the role of androgens in regulation of angiogenesis is still not fully understood [5, 6]. Further, most investigations have focused on the initial vascular response following ADT, typically from a few hours up to one week. The more long-term effects of ADT on tumor vasculature may be equally or more important in understanding PC progression, and for selection of combined treatments.

Quantitative dynamic contrast-enhanced magnetic resonance imaging (DCE MRI) is a promising non-invasive imaging modality for assessing tumor vacularization and treatment responses [7]. Conventional extracellular contrast agents rapidly distribute throughout the blood plasma and extravasate into the extracellular extravascular space (EES) after being intravenously (i.v.) injected. These agents affect the tissues’ relaxation rates, causing increased signal intensity on T1-weighted MR images. The signal intensity changes versus time reflect vascular features and may be analyzed directly using semi-quantitative approaches or quantitatively by pharmacokinetic models. Extracted image parameters enable estimation of the tumors’ vascular characteristics and their angiogenic responses to therapeutic interventions, potentially also serving as indirect non-invasive biomarkers of tumor hypoxia. However, the use of DCE MRI parameters is dependent on correct tumor physiological interpretations. The current understanding is still incomplete and to a large extent based on assumptions yet to be validated. Therefore, the present study also interpreted how the DCE MRI parameters we quantified correlated to the underlying biology, by performing a quantitative analysis of fluorescent immunohistochemistry (qIHC) of excised tumor tissue.

Thus, this study aimed to characterize the vascular responses to RT and ADT using an experimental, androgen-sensitive PC model. Animals were subjected to in vivo DCE MRI and analyzed with both semi-quantitatively and by pharmacokinetic models. The animals’ tumors were excised immediately after MRI for quantitative ex vivo analysis of tumor vascularization (9 F1), perfusion (Hoechst 33342), and hypoxia (pimonidazole).

Materials and methods

Experimental tumor model

Tumors were generated by subcutaneous (s.c.) implantation of (~2 mm)3 tumor tissue from the human, androgen-sensitive CWR22 PC xenograft, together with a 12.5 mg sustained-release testosterone pellet (Innovative Research of America, Sarasota, FL), into adult male Balb/c nude mice. These pellets ensure stable, human serum testosterone levels and contribute to improved recapitulation of androgen-regulated homeostasis [8]. Animal experiments were performed in accordance with protocols approved by the institutional animal care and use committee, in compliance with guidelines on animal welfare of the Norwegian National Committee for Animal Experiments. Tumor sizes were measured using calipers from implantation until the end of the experiment.

Treatments and experimental groups

The study included two consecutive experiments with untreated (Ctrl), irradiated (RT), androgen-deprived (AD), and castration-resistant (CR) tumors. The CWR22 xenograft mimicks the clinical situation of PC disease progression, by regressing after ADT, and showing stability during long-term ADT before recurrent CR tumor growth is evident, a disease which presently is incurable in the clinic. To allow tumor size-independent comparisons all analyzed tumors had diameters of 8 mm.

Firstly, animals in the Ctrl group (n = 4) were subjected to DCE MRI when the tumor diameter reached 8 mm. For the RT group (n = 6), tumors reaching 8 mm in diameter were irradiated with a single dose of 15 Gy, followed by DCE MRI 24 h later, a time-point selected based on a previous study investigating radiation-induced effects in the CWR22 xenograft [9]. Radiation was delivered using a 60Co source (Mobaltron 80, TEM Instruments, Crawley, UK), with a dose rate of 0.8 Gy/min. Tumor irradiation was performed by placing the tumors in the corners of a 10 × 10 cm radiation field, keeping the animals’ bodies outside the field to minimize radiation dose deposition to normal tissues. ADT was performed when the shortest tumor diameter reached 12 mm, by surgically castrating the animals and removing the testosterone pellet. One month post-castration the tumor size was reduced to 8 mm in diameter, upon which the animals were subjected to DCE MRI (n = 4). Following MRI all tumors were excised and immediately snap-frozen for qIHC.

Secondly, the experiment was repeated to analyze Ctrl (n = 5), AD (n = 5), and CR (n = 5) tumors of identical size as in the first experiment. All tumors were subjected to DCE MRI, and blood sampling was performed for measuring serum prostate-specific antigen (PSA) levels.

Radiation, castration, and MRI were performed under anesthesia, after s.c. injections of a mixture of 2.4 mg/ml tiletamine and 2.4 mg/ml zolazepam (Zoletil vet, Virbac Laboratories, Carros, France), 3.8 mg/ml xylazine (Narcoxyl vet, Roche, Basel, Switzerland), and 0.1 mg/ml butorphanol (Torbugesic, Fort Dodge Laboratories, Fort Dodge, IA), diluted 1:5 in sterile water, in doses of 5 μl/g prior to radiation, and 7.5 μl/g before MRI acquisitions and castration. Analgesia was provided to castrated animals by s.c. injections of buprenorphine (Temgesic, Schering-Plough, Brussels, Belgium) in a dose of 0.1 mg/kg.

Prostate-specific antigen

Blood samples were withdrawn from the animals’ femoral veins immediately before euthanasia and tumor excision. The samples coagulated before being centrifuged and frozen at −80°C until all samples were analyzed simultaneously. Serum PSA levels were measured using the fluoroimmunometric AutoDELFIA ProStatus PSA Free/Total kit (PerkinElmer Life and Analytical Sciences, Wallac Oy, Turku, Finland).

DCE MRI acquisition and analysis

DCE MRI was acquired as previously described [10], and analyzed using in-house developed software in IDL (Interactive Data Language v6.2, Research Systems Inc., Boulder, CO), as illustrated in Figure 1. For the central slice of each tumor, a region-of-interest (ROI) was manually traced in T1-weighted DCE MR images, excluding surrounding skin and connective tissue. Firstly, semi-quantitative analysis was performed. The time-dependent relative signal intensity (RSI(t)) was calculated for each image voxel using the equation RSI(t) = (SI(t) – SI(0))/SI(0), where SI(0) refers to the pre-contrast signal intensity and SI(t) the post-contrast signal intensity in the voxel at time t. The semi-quantitative parameter area under the RSI(t) curve ( AUC(t)) was calculated for t = 1, 2, 3, 4, 5, 10, 15, and 20 min.
Figure 1

Dynamic contrast-enhanced MRI of experimental prostate cancer. Following dynamic contrast-enhanced (DCE) MRI the tumors were traced in the T1-weighted images. Firstly, semi-quantitative analysis was performed on the time-dependent relative signal intensity (RSI(t)) curves, by calculating the area under the RSI curve (AUC(t)), of each tumor voxel, for different time-points. Secondly, pharmacokinetic analysis was performed using the Tofts model, producing parametric tumor maps of Ktrans, k ep , and V e . Examples of parametric tumor maps are shown to the right.

Secondly, pharmacokinetic analysis was performed using the two-compartment Tofts model [11]. Signal intensities were converted into contrast agent (CA) concentration (C(t)) by using the method of Hittmair [12]. The vascular input function (VIF) needed in Tofts modeling has previously been established for our male mouse strain: VIF = 3.57 e-(0.025)t + 1.45 e-(0.0074)t[10]. Using the VIF parameters and the CA concentration curves, each voxel was fitted to the Tofts model, allowing estimations of the forward volume transfer constant of CA from blood plasma into the EES, Ktrans (s-1), the backward transfer constant from EES to blood plasma, k ep (s-1), and the fractional distribution volume of CA, V e (a. u.), as derived from Ktrans/k ep . When performing Tofts modeling, the fit fails to converge for some voxels due to low CA uptake or voxels providing negative values. These unfitted voxels were set equal to 0 and included in calculation of parametric tumor maps. The unfit fraction; i.e. the sum of unfitted voxels versus the total number of voxels within the tumor ROI, was monitored to investigate the possible relation between this fraction and tumor necrosis assessed in the tissue sections.

Sixty minutes prior to euthanasia, 80 mg/kg of pimonidazole hydrochloride (1-[(2-hydroxy-3-piperidinyl)propyl]-2-nitroimidazole hydrochloride, Natural Pharmacia International Inc., Burlington, MA, USA) in 0.5 ml saline was injected intraperitoneally (i.p.) in the animals to determine tumor hypoxia. Tumor perfusion was found by i.v. injection of 15 mg/kg Hoechst 33342 (Sigma-Aldrich, Oslo, Norway) in 0.1 ml saline 5 min before euthanasia. Immediately after dissection, tumors were snap-frozen in liquid nitrogen and stored at −80°C until being shipped to the Department of Radiation Oncology, Radboud University, Nijmegen Medical Center, Nijmegen, The Netherlands, for staining and imaging. The central slice of the tumor was cut before being stained and scanned according to previously described procedures [13], to obtain three spatially matched immunofluorescent images of tumor vascularization (9 F1; TCL image, TNO, Delft, The Netherlands), tissue perfusion (Hoechst 33342), and hypoxia (pimonidazole).

Quantitative analysis of immunohistochemistry

Spatial maps of hypoxia, vessels, and perfused area were created from immunofluorescent images. Firstly, hypoxic regions were determined from pimonidazole stains using MATLAB 7.0.1 (The MathWorks, Natick, MA). Different methods were used due to differences in signal intensity and image contrast between different batches. For nine cases: (a) the image was digitally filtered to correct for unequal illumination in each tile, (b) a Gaussian blur was applied to smooth the image (σ = 8 px), and (c) the threshold was set to the mean of the 5th and 95th percentile intensity value. For six cases the threshold was set to 0.3 × 95th intensity percentile, and in one case the threshold was set manually. Secondly, vessels were manually delineated in 9 F1 stains. Thirdly, perfusion was manually segmented using local and/or global thresholds in Hoechst stains. The following regions were manually delineated based on a combination of stains: (a) gross tumor region, (b) artifacts, (c) necrosis; regions with a strong background signal from 9 F1 and a low signal from pimonidazole, (d) other tissue; tumor regions with markedly different texture in 9 F1 and pimonidazole stains, and a defined transition to viable tumor regions. Two additional tumor regions were defined: (e) net tumor; gross tumor excluding artifacts, and (f) viable tumor; net tumor excluding necrosis and other tissue. The necrotic fraction ( NF) was defined as necrotic area/net tumor area.

A vessel was defined as an independent contiguous 8-connected region in the vessel map, and lumens were considered part of the vessel. The following vessel parameters were extracted: total number of vessels (VN), vessel density ( VD) as VN/viable tumor area, vascular area fraction ( VF) as vascular area/viable tumor area, and mean vessel size ( VS).

A vessel was considered to be perfused if it was less than 20 μm away from a perfusion stain with at least 10% of its area. The following perfusion parameters were extracted: perfusion stain fraction (PF) as perfusion stain area/viable tumor area, and perfused vessel fraction ( PVF) as number of perfused vessels/ VN.

These parameters measured the degree of hypoxia: hypoxic fraction (HF) as hypoxic area/viable tumor area, and mean distance from hypoxic pixels to nearest vessel ( DHV). Since pimonidazole staining intensity could not be converted to oxygen pressure (pO2), the exact boundary between hypoxia and normoxia cannot be decided; HF and DHV are considered to be estimates proportional to true values. The theoretical normoxic fraction was calculated based on the assumption that all (perfused) vessels supply oxygen and that the diffusion range of oxygen is 70 μm: fraction of viable tumor tissue < 70 μm away from a vessel ( VF70V).

Estimation of acute hypoxia was based on the following assumptions: (a) since transport of Hoechst requires a functional and oxygenated vascular network, areas with perfusion staining were considered functional after Hoechst injection; (b) areas of hypoxia staining were considered to reflect hypoxia after pimonidazole injection. By assuming that the metabolic activity was sufficiently low to not affect the diffusion distance of oxygen, it was further assumed that the diffusion distance of oxygen was longer than the diffusion distance of Hoechst, and thus, that the amount of pimonidazole staining within a 50 μm distance from perfusion stains was proportional to the amount of acute hypoxia. The proportion of the total HF that was acute was named “acute hypoxia score” ( AHS). The remaining proportion is thus representing chronic hypoxia (= 1 – AHS).

The manual delineations and thresholds were performed in Gimp 2.6.4 (, and quantitative analysis in an in-house program written in Python 2.6.6 (, with the scipy extension (

Statistical analysis was performed using PASW Statistics 18.0 (IBM, Somers, NY). Differences were analyzed using two-sided t-tests and correlations assessed by Pearson’s correlations ( r). A significance level of 5% was used.

Assessment of response to radiotherapy and androgen-deprivation therapy by quantitative immunohistochemistry

To characterize the response to RT and ADT, qIHC was performed. Matching sets of images of tumor hypoxia (pimonidazole), endothelium (9 F1), and perfusion (Hoechst 33342) (Figures 2A–C), as well as a combined image (Figure 2D), were obtained. Corresponding maps of the tumor-specific features (Figures 2E–H) were extracted and used in qIHC.
Figure 2

Fluorescent immunohistochemical images and maps. Tumors were triple-stained with fluorescent immunohistochemical markers of hypoxia (pimonidazole), endothelium (9 F1), and perfusion (Hoechst 33342), to produce images of hypoxia ( A), endothelium ( B), perfusion ( C), and a combined image ( D). Corresponding maps were extracted and used in the quantitative analysis ( E-G). Composite map where artefacts and necrosis, which were excluded from the measurements, are shown in white ( H).

Figure 3 shows the results summarized per experimental group. Compared to Ctrl tumors, AD tumors presented significant increases in several vascular qIHC parameters; a 3.5-fold increase in VN ( p = 0.012) (Figure 3A), a 5.1-fold increase in VD ( p = 0.009) (Figure 3B), and a 3.2-fold increase in VF ( p = 0.003) (Figure 3D). The viable tissue was located closer to the vasculature in AD tumors compared to in Ctrl tumors, as reflected by a 1.7-fold increase in VF70V ( p < 0.001) (Figure 3G). Also the hypoxic stains were closer to vessels in AD tumors, reflected by DHV being 0.7-fold decreased ( p = 0.002) (Figure 3H) compared to Ctrl tumors. AD induced an insignificant decrease in VS (Figure 3C) and NF (Figure 3I). The changes in PF (Figure 3E) and VPF (Figure 3F) in AD tumors compared to Ctrl tumors were statistically insignificant; yet they indicate that tumors regressing in size after ADT acquire more vessels at a higher density, and remain equally perfused and highly functional.
Figure 3

Quantitative immunohistochemical analysis. Intergroup differences in parameters from quantitative immunohistochemical analysis of triple-stained tumor tissue sections (9 F1; endothelium, Hoechst 33342; perfusion, pimonidazole; hypoxia) from untreated (Ctrl), irradiated (RT), and androgen-deprived (AD) prostate carcinoma (PC) xenografts, presented as mean and s.e.m. per group. Significant differences ( p < 0.05) compared to Ctrl tumors are indicated with a *.

Following RT, the sole significant vascular change compared to Ctrl tumors was the 0.4-fold decrease in PF ( p = 0.031) (Figure 3E). The changes in VN (Figure 3A), VD (Figure 3B), VS (Figure 3C), VF (Figure 3D), DHV (Figure 3H), and NF (Figure 3I) were insignificant. The other parameters (VPF, VF70V) remained unchanged.

Generally, these results unveiled minor radiation-induced effects on tumor vasculature, whereas AD tumors showed significantly increased tumor vascularization. Compared to the Ctrl cohort, AD tumors developed many small vessels covering a significantly larger fraction of the total tumor area. This increased tumor vascularization amongst AD tumors is visualized by perfusion and endothelium maps in Figure 4.
Figure 4

Tumor vascularization in androgen-deprived versus untreated tumors. Increased tumor vascularization in an androgen-deprived (AD) tumor (left column) compared to untreated (Ctrl) tumor (right column) visualized by combined perfusion and endothelium maps ( A and D, respectively), and separate maps of perfusion ( B and E, respectively) and endothelium ( C and F, respectively).

The HF did not differ significantly between the groups (Figure 3J). However, independent of HF the estimated AHS was 2.5-fold higher ( p = 0.002) in AD tumors compared to in Ctrl tumors, whereas RT tumors only showed an insignificant increase (Figure 3K).

Assessment of response to radiotherapy and androgen-deprivation therapy by dynamic contrast-enhanced MRI

Figures 5A-D show that inter-group differences in AUC values were most evident at initial time-points and less pronounced at later time-points. Compared to Ctrl tumors, RT induced insignificant changes in AUC values, whereas AD tumors presented significantly higher AUC values than Ctrl tumors at all analyzed time-points, with the largest increase at the initial time-point, AUC 1 min, being 2.8-fold (p = 0.016). The AUC values remained high in AD tumors during the initial time-points before gradually decreasing relative to Ctrl tumors, the difference being 2.5-fold at 2 min ( p = 0.007), 2.3-fold at 3 min ( p = 0.004), 2.0-fold at 4 min ( p = 0.002), 2.0-fold at 5 min ( p = 0.002), 1.7-fold at 10 min ( p = 0.012), 1.6-fold at 15 min ( p = 0.020) and 1.5-fold at 20 min ( p = 0.034).
Figure 5

Semi-quantitative and pharmacokinetic analysis of dynamic contrast-enhanced MRI. Both semi-quantitative and pharmacokinetic DCE MRI parameters unveiled increased vascularization in androgen-deprived (AD) tumors. Intergroup differences between untreated (Ctrl), irradiated (RT), and androgen-deprived (AD) prostate carcinoma (PC) xenografts for the semi-quantitative parameter area under the relative signal intensity curve ( AUC) (a.u.) for 1 min ( A), 5 min ( B), 10 min ( C), and 20 min ( D), and Tofts kinetic model parameters Ktrans (s-1) (E), k ep (s-1) (F), and V e (a.u.) (G), in addition to the fraction of unfitted voxels (H), summarized as mean and s.e.m. per group. Significant differences (p < 0.05) compared to Ctrl tumors are indicated with a *.

In Figures 5E-G, intergroup differences in Tofts pharmacokinetic parameters are shown. Compared to Ctrl tumors, RT induced a 0.5-fold reduction in Ktrans, whereas a significant 1.8-fold increase was unveiled in AD tumors (p = 0.041) (Figure 5E). For k ep , RT tumors showed a 0.8-fold reduction (p = 0.002), in contrast to a 1.5-fold increase in AD tumors, when compared to Ctrl tumors (Figure 5F). Large intra-group differences in V e resulted in insignificant changes in both RT and AD tumors (Figure 5G). The unfit fraction showed a 3.0-fold increase (p = 0.026) in RT tumors, and a 0.6-fold reduction ( p = 0.046) in AD tumors, compared to Ctrl tumors (Figure 5H).

Correlations between quantitative immunohistochemistry and dynamic contrast-enhanced MRI

Figure 6 shows resulting Pearson correlation coefficients (r) from correlating parameters from DCE MRI and qIHC. Initial AUC values (< 4 min) correlated very strongly and positively ( r = 0.8–1.0) to VD and VF, whereas correlations at later time-points were less pronounced ( r = 0.6–0.8). The Tofts parameters Ktrans and k ep also correlated significantly to VD, although not as strong as the AUC values ( r = 0.6–0.8). Tumor perfusion, as reflected by PF, was weakly correlated to AUC values ( r = 0.4–0.6). Of note, VS correlated only insignificantly, and negatively, to all AUC values. NF was negatively correlated to initial AUC values ( r = 0.4–0.6). The overall HF did not correlate to any AUC parameter, but DHV correlated negatively ( r = 0.6–0.8) to all AUC values obtained from 1 min to 10 min. The AHS fraction correlated positively and very strongly to NF ( r = 0.85). Tofts parameters correlated poorly to HF, but k ep was strongly positively correlated to AHS (r = 0.72).
Figure 6

Correlations between parameters from dynamic contrast-enhanced MRI and quantitative immunohistochemistry. Correlations between DCE MRI parameters from ( A) semi-quantitative and ( B) Tofts kinetic analysis, and qIHC parameters. Color labeling reflects correlation strength (Pearson correlation coefficients), as indicated in the color scale (top). Positive and negative correlations are indicated with + and –, respectively, and encircled + and – denotes significant correlations ( p < 0.05). Abbreviations: AUC; area under the relative signal intensity ( RSI) curve, VS; mean vessel size (μm2), VD; vessel density (number of vessels/mm2), VF; vessel area fraction, PF; perfused area fraction, DHV; distance from hypoxia to vessels (μm), NF; necrotic fraction, HF; hypoxic fraction, AHS; acute hypoxia score\.

Vascular changes occurring in the development of castration-resistant disease

The large vascular changes identified in AD tumors motivated a repeated experiment including tumors re-growing in the CR disease state. Figure 7A visualizes the development of CR tumors 5.4 ± 0.4 months after ADT by castration. Figure 7B shows the expression of serum PSA in blood samples from animals with Ctrl tumors, AD tumors, and CR tumors. The reduction in serum PSA from Ctrl to AD was significant (p = 0.032), whereas the rise in PSA from AD to CR was close to significant ( p = 0.085). The DCE MRI parameters best reflecting tumor vascularization, as identified from Figure 6, were calculated from the Ctrl, AD, and CR tumors. In Figure 7C, an increase in AUC 1 min and AUC 2 min from Ctrl tumors to AD tumors was again seen (1.8-fold increase, p = 0.011 and 1.7-fold increase, p = 0.025, respectively). The transition from AD tumors to CR tumors unveiled a further increase in tumor vascularization, as seen by the 1.4-fold increase ( p = 0.010) in AUC 1 min and a further 1.3-fold increase ( p < 0.001) in AUC 2 min. Also the Tofts parameters in Figure 7D showed similar pattern; a 1.3-fold increase (p = 0.012) in Ktrans from Ctrl tumors to AD tumors, and a further 1.4-fold increase (p = 0.043) from AD tumors to CR tumors, whereas k ep showed an insignificant change from Ctrl tumors to AD tumors, followed by a significant 1.6-fold increase (p = 0.007) from AD tumors to CR tumors.
Figure 7

Vascular changes in the transition from untreated (Ctrl), via androgen-deprived (AD), to castration-resistant (CR) disease. Response to androgen-deprivation therapy (ADT) on tumor volumes, showing that CR tumors developed 5.4 ± 0.4 months after ADT ( A). Response to ADT on serum prostate-specific antigen (PSA) expression measured in blood samples withdrawn from the animals ( B). The vascular parameters AUC 1 min and AUC 2 min calculated from semi-quantitative analysis of DCE MRI ( C). The vascular parameters Ktrans and k ep derived from Tofts pharmacokinetic modeling of DCE MRI (D). Significant differences ( p < 0.05) compared to Ctrl tumors are indicated with a *.

In the present study the vascular responses to RT and ADT in experimental PC were characterized by qIHC and DCE MRI. Our key findings were the increased tumor vascularization following ADT, and the identification of a set of DCE MRI parameters non-invasively reflecting these vascular changes.

The xenograft used was originally derived from a patient’s primary, androgen-sensitive PC that later developed metastatic disease [14], thus being a clinically relevant model of advanced PC. This model enables investigations of the response to different interventions, in particular ADT, since this xenograft regresses after castration and shows stability before recurrent CR growth is evident, as also experimentally verified in this study (Figures 7A-B).

By qIHC an increased tumor vascularization was detected in AD tumors one month post-castration, compared to Ctrl tumors. This was reflected by the increased VN, VD and VF, although VS was smaller (Figure 3). Further, the vessels appeared to be highly functional despite their smaller size. This seems possible since the vessels were equally, or even more perfused as compared to Ctrl tumors, as reflected by unchanged VPF and the clear tendency of increased PF (Figure 3). Together, these findings are consistent with tumor neovascularization following ADT. We found a small, but insignificant, increase in HF. However, more interestingly the DHV was significantly shorter in AD tumors compared to in Ctrl tumors, which encouraged a further analysis of whether ADT had altered the ratio of acute versus chronic hypoxia independently of the total HF. For this purpose we estimated AHS (Figure 3K), revealing increased acute hypoxia, and thus less chronic hypoxia, in AD tumors compared to Ctrl tumors.

The mechanisms responsible for the ADT-induced changes in tumor vascularization and hypoxia are presumably caused by a complex interplay of different processes. First, a substantial increase in vessel formation may be expected during unperturbed growth from 8 mm (the diameter of tumors in Ctrl tumors) to 12 mm (the diameter of tumors at the time of castration). Following castration, volume reduction caused by death of androgen-sensitive, endothelial cells occur, likely resulting in vessel shrinkage. However, our results indicate a very high number of functional vessels with capability to transport blood, as reflected by the preserved tumor perfusion. Moreover, we observed an increased VD in AD tumors. One month after castration the tumor volume regressed by approximately a factor 3 (from a tumor volume of ≈ 864 mm3 (diameter of 12 mm) to a tumor volume of ≈ 256 mm3 (diameter of 8 mm)). A corresponding 3-fold increase in VD would thus be expected if the number of vessels remained identically as pre-ADT. However, the observed increase in VD was of a factor 5, and hence, a high number of vessels with smaller VS may not be explained by ADT-induced volume reductions alone. It has previously been shown that an immediate effect of ADT involves induction of acute hypoxia, including activation and stabilization of the hypoxia-inducible factor 1α (HIF-1α), and subsequent expression of HIF-1α target genes, such as the vascular endothelial growth factor (VEGF) [1517], a key player involved in tumor neovascularization. Given that this occurs, formation of large numbers of small, new vessels may be expected. Such a neovascularization may explain the significant changes detected in vascular and perfusion-related qIHC parameters.

The AHS increased in AD tumors compared to in Ctrl tumors. Since perfusion qIHC parameters increased in AD tumors compared to in Ctrl tumors, it is not likely that the vessels are perfusion-limited or occluded, as is expected by the increased presence of acute hypoxia. Yet, small fluctuations in blood flow may result in temporary occlusions and transient hypoxia. However, such occlusions do not seem to severely have affected tumor vascularization and perfusion in AD tumors. In fact, it appears as if the vascular function in AD tumors is superior to in Ctrl tumors. It could be that the increase in acute hypoxia is followed by a reoxygenation of the tumor tissue. If extrapolating to the clinical situation, such a reoxygenation hypothesis may provide a possible explanation as to why neoadjuvant ADT combined with RT has been shown to improve treatment outcome and patient survival [2, 3]. However, it has also been shown that reoxygenation after acute hypoxia contributes to increased genomic instability, and thus disease progression [18], including increased formation of metastasis [19, 20].

The increased vascularization following ADT may also relate to development of a more aggressive disease, and hence, represent a step in the transformation into CR disease. Clinical studies in PC have previously connected microvessel density to poor survival [2123]. When we repeated the experiment and extended it to also include CR tumors, where the serum PSA levels increased and renewed growth appeared, we detected an even further increased tumor vascularization compared to both Ctrl tumors and AD tumors (Figure 7C and D). Highly vascularized tumors are usually well oxygenated and sensitive to radiation. These tumors may, paradoxically, have enhanced activation of angiogenic factors normally found in hypoxic, radiation resistant tumors and in tumors associated with a poor prognosis [24]. Both for breast cancer and colorectal cancer correlations between enhanced expression of angiogenic factors and poorer prognosis and shorter disease-free survival have been shown [25, 26]. Moreover, highly angiogenic tumors are more likely to develop metastatic disease as the probability of secreting tumor cells into the circulation is higher [27].

Human studies characterizing vascular effects following ADT are limited. Animal studies have shown that immediate responses to ADT involve blood flow reductions and vascular degenerations [2831]. The recent study by Godoy et al.[6] shows vascular recovery after ADT, as demonstrated by increased microvessel density and vascular lumen diameter. The difference in the study by Godoy et al. to previous studies is that they not only characterized the immediate effect of ADT on tumor vasculature, but prolonged their post-ADT investigation period. Thus, these results indicate that ADT induces an immediate, transient degeneration of tumor vascularization before androgen-sensitive, endothelial cells develop alternative mechanisms to reproduce the vascular network in the absence of androgens. A recent DCE MRI study in PC patients oppositely demonstrated decreased Ktrans after 1 and 3 months of ADT [32]. One explanation for the different results may be related to the timing of the measurements. The clinical study may comply with the preclinical results of initial vascular degenerations. Our results, obtained 1 month post-ADT, anticipated to correspond to about 5 months in humans after accounting for the five times faster metabolism in mice, may reflect a later disease stage with vascular recovery, and comply with the results by Godoy et al.[6]. The challenges with translating time-dependent treatment effects in xenografts to humans indicate that studies characterizing the long-term effects of ADT on tumor vascularization should be further investigated in the clinical setting. One approach could be to perform DCE MRI also at later disease stages after ADT, preferably as repeated measurements in the same patients.

The results from qIHC following RT showed results being consistent with previous findings. RT did not result in substantial effects on VD and VF 24 h post-irradiation, only an insignificant increase in vessel size VS was found. The increased VS may relate to radiation-induced dilatation of tumor capillaries, as has been previously shown [24, 33]. Further, RT resulted in a significant decrease in PF, as also shown by others [34]; this is likely attributed to the formation of radiation-induced edemas and capillary injuries. Successful RT results in irreversible DNA damages, which are in accordance with the increased NF in the RT tumors. Moreover, it is also previously shown that vascular alterations after RT can be detected by DCE MRI [35].

Vascular qIHC parameters, like VD and VF, correlated strongly to AUC values from the initial time-points (Figure 6). Moreover, AUC values were negatively correlated to DHV. This may be related to the higher fraction of viable cells, as reflected by the VF70V parameter, close to the vessels in tumors with short DHV. Applying the Tofts kinetic model, the k ep parameter improved the correlation to VS compared to using AUC values. VD was also positively correlated to the Tofts parameters, albeit less than the AUC values. In summary, the correlations between DCE MRI parameters and vascular qIHC parameters showed that AUC values reflect VD and VF strongly, whereas estimation of VS from DCE MRI requires the use of the Tofts kinetic model. Since parameters from the Tofts model previously have shown to be indirectly related to tumor hypoxia [7, 36], the poor correlations between HF and all DCE MRI parameters were surprising. It should be noted that the HF was not found to correlate to other qIHC parameters either, nor differentiate treatment groups in the t-test. One possible reason may be that the analyzed tumors, with a few exceptions, showed relatively similar HF, and hence, the spread in the data were too low to resolve any such correlation. By estimating AHS (Figure 3K), substantially higher and significantly positive correlations to several DCE MRI parameters were found (Figure 6).

The NF was strongly correlated with the unfit fraction from the Tofts model, i.e. the voxels with low contrast enhancement. Bradley et al.[37] and Galbraith et al.[38], as well as our own previous study [10], have revealed similar correlations, providing support for the unfit fraction as a non-invasive biomarker of tumor necrosis.

In this study, the potential of DCE MRI to provide non-invasive biomarkers of tumor vascularization was assessed. After the recognition of the importance of the condition of the tumor vasculature, including tumor hypoxia, for treatment response and disease outcome in oncology [39, 40], several functional imaging modalities have been investigated for their ability to provide such non-invasive biomarkers. In addition to DCE MRI and diffusion-weighted (DW) MRI, positron emission tomography (PET), dynamic computed tomography (CT), and ultrasound Doppler imaging, have been proposed as promising modalities. The benefits with MRI include that it is a wide-spread, clinically established modality, also in advanced PC, and hence, any new sequences, such as DCE MRI, may easily be added. Moreover, compared to PET and CT, MRI benefits from the avoidance of ionizing radiation exposure, as well as being more cost-effective.

In conclusion, DCE MRI non-invasively detected an increased and highly functional vascular network in experimental prostate tumors after ADT, which was confirmed by qIHC. The findings encourage further studies into whether these vascular changes are beneficial for combined RT, or if treatment with anti-angiogenic therapy or vascular disrupting agents may be a strategy to improve the therapeutic efficacy of ADT in advanced PC.

We thank F. Saatcioglu and Gunhild Mælandsmo for providing the CWR22 xenograft. The technical assistance with animal experiments provided by Alexandr Kristian is greatly acknowledged.

Grants received from the South-Eastern Norway Regional Health Authority (grant 2009070, to KR) and the European Union 7th Framework Programme (grant 222741 – METOXIA).

Authors’ Affiliations

Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital
Institute of Clinical Medicine, University of Oslo
Department of Oncology, Akershus University Hospital
Department of Physics, University of Oslo
Department of Radiation Oncology, Radboud University Nijmegen Medical Center
Department of Human Oncology, University of Wisconsin
Prostate Molecular Oncology Research Group, Academic Unit of Clinical and Molecular Oncology, Institute of Molecular Medicine, St. James’s Hospital and Trinity College Dublin
Faculty of Mathematics and Natural Sciences, University of Bergen

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© Røe et al.; licensee BioMed Central Ltd. 2012

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Saturday, February 9, 2013

Fascinating comment on the Lucy Lawless 'incident'

Jenny@The Standard has this to say:

9 February 2013 at 9:10 am

That Lucy Lawless and others like her are doing a “Community Service” is undoubtable. That this community service will go unrecognised and even punished by the state to a disproportionate degree can not be doubted either.

This is why:

In a statement, Shell Todd Oil Services general manager Rob Jager said the company had always supported police’s response to the incident.

“That extended to supporting the police’s case for reparation.

NZ Herald

Public lobbying from the oil company of the Police, for their over the top action, in trying to intimidate the protesters with “excessive” “unjustified” and disproportionate, to the ‘crime’, damages.

The hugely disproportionate damages sought by the police for on the face of it minor trespass charges, is a political decision, clearly more in line with the interests of the oil company than with natural justice.

That the decision to seek such extraordinary punitive damages was taken by the police points to an increasing politicisation of our police force.

Luckily this politicisation has not extended to the judiciary, and the police have failed with their campaign of intimidation in court.

But will the police in the heeding of the oil company, now turn to more open and direct forms of “excessive” and “unjustified” intimidation of environmental protesters outside of court?

The next time the oil companies take measures to destroy the biosphere for personal gain, and citizens take it on themselves to peacefully impede them. Will the police acting from political motives in line with the wishes of the polluters respond disproportionately?

Those who made this decision need to be questioned.

Because going on their record in court, the chances are very high, that these same police commanders will be ordering over the top actions against any future such peaceful protests.

In this event, I don’t think we will seeing any such outrageous public statements similar to the one from the oil company we saw yesterday.

In a statement, Hell Toad Oil Services general manager Job Rager said the company had always supported police’s response to the incident.

“That extended to supporting the police’s case for retribution.

HZ Nerald

The point of the above comment is to reveal that extreme and dirty tactics will be used by the police to protect what Naomi Klein et al have called a “rogue industry and its lobby”.

The public statement of support for the police’s extreme demands for reparation against protesters trying to protect the Arctic. Is a rare example of public lobbying, of the forces of the state by an oil company.

Most of this lobbying of the police and the state is usually done behind closed doors.

The oil companies’ have a huge and well funded lobbying machine that puts out feelers, to government, to political parties, the civil service, and yes even the police and other state forces. (witness the use of the navy against Greenpeace and Te Whanau a Apanui) Through well funded lobbying, that usually goes on behind closed doors, in private, with access generally not available to the public, the polluters are demanding that extreme measures be taken against those who dare to oppose their interests. And as we can see in this case, their demands are being answered.

Rarely does this lobbying break out into the public arena. And despite the failure of the police’s efforts to intimidate the protesters in court, I imagine that a big thank you cheque will be finding its way to some police welfare organisation, or favourite charity, as an encouragement to keep up the good work.

1 comment:

Paranormal said...

Molehill meet mountain.

'Lobbying' is not the correct word in this case. Any commercial operation will seek reimbursement of lost costs when the equipment they have significant capital invested in is unable to pay it's way through criminal behaviour.

It's more normally called reparation and is requested through the police every day by both individuals and corporates who have been victims of criminal activity.

Lawless admitted her guilt of a criminal actrion.

The real 'lobbying' is a political beat up by you and your Green colleagues.


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Mitt Romney Lands Second $100 Million Month, Pres. Obama & DNC Raise $75 Million In July

Source: CNN

The fundraising effort of presumptive Republican presidential nominee Mitt Romney brought in more than $100 million for the second month in a row, his campaign said Monday morning.

The $101.3 million haul is just shy of the campaign’s June fundraising, which reached $106.1 million.

Romney’s effort ended July with $185.9 million cash on hand, up from the $160 million in the campaign war chest at the end of June.

President Barack Obama and the Democratic National Committee, meanwhile, raised $75 million in July, marking the third consecutive month the president has been outraised by his Republican challenger. Obama’s similar alliance of committees raised $71 million in June.

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[Mongrel] Mongrel::HttpHandlerPlugin

Rick Olson technoweenie at gmail.com
Thu Oct 19 20:03:48 EDT 2006

On 10/19/06, Zed A. Shaw <zedshaw at zedshaw.com> wrote:
> On Fri, 20 Oct 2006 08:24:13 +1000
> "Carl Woodward" <cjwoodward at gmail.com> wrote:
> > Hi,
> >
> > I'm trying to write a file upload monitor for mongrel that uses
> > juggernaut to alert the browser of a change. I'm writing a plugin that
> > uses Mongrel::HttpHandlerPlugin but it looks like the only method that
> > gets called is process and that is only when the upload has finished.
> > I'm setting the following:
> >
> > @request_notify = true
> >
> > But I am still only getting process to come through.
> >
> > Any ideas?
> Did you check out the existing mongrel_upload_progress code works?  I'd base your work on that before striking out on your own.

It should work just fine.  You just need to write a custom action to
return the status for juggernaut though.  I'm not sure how you prefer
to do that.  The MUP example in the docs show a rails action to return
custom javascript code for an iframe...

Rick Olson

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Video: Breathing Exercises For Relaxation

In this video Sage Rountree explains how breathing has a direct physical effect on your mind and body. She also offers examples of exercises you can use to feel calmer and more relaxed before races and hard workouts–portable recovery tools that can be very powerful.

RELATED VIDEO — Yoga For Runners: Breathing

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Alan Webb, Version 2.0

Alan Webb training in Albuquerque as a young pro in 2003. Photo:

Chasing Perfection

Widely considered one of the most purely talented American runners, Webb trained on his own much of his career because no one in the country could keep up with him. As a teenage phenom, he famously set the high school mile record of 3:53.43 in 2001. He spent only one season at the University of Michigan under legendary coach Ron Warhurst, much of which became fodder for the book, “Sub-4:00: Alan Webb and The Quest for the Fastest Mile,” before returning to his high school coach Scott Raczko and turning pro at age 20.

He went on to make the 2004 U.S Olympic team in the 1,500 meters (the mile’s metric counterpart) and continued his progression through 2007, when at the age of 24, he broke Steve Scott’s long-standing American mile record with a 3:46.91 effort in Belgium. That mark remains the eighth-fastest in history, ranking him slightly ahead of Bernard Lagat and Sebastian Coe on the all-time list.

Webb’s performances and continued progress during that six-year stretch were surreal, but instead of being untouchable, he remained a people’s champ. At 5-foot-9 and 145 pounds, he didn’t look unnaturally thin and admitted to a sweet tooth. He raced from the front, fueled by equal parts laser focus and cold-blooded anger. And he showed his emotion, often finishing a big race by flexing his muscles for the crowd.

“When I first saw him at meets, I was always impressed that he would be the last person to leave the track,” Ray Flynn, Webb’s longtime agent, recalls of Webb’s meticulous cool-downs. “I was secretly impressed that he was doing everything he possibly could to be better.”

But something happened on his way to the top. Webb never truly ascended the world order as many had expected. Although he was eliminated in the first round of the 2004 Olympics in Athens (his first global championship), he advanced to the world championship finals in 2005 and 2007, placing sixth and eighth, respectively.

Then his career took an improbable and unexpected turn. Webb raced poorly and inexplicably missed the 2008 Olympic team. Over the next three years, things got progressively worse. Amid nagging pains, more coaching changes and so-so results, he prematurely moved up to the 5,000-meter run and didn’t make it to the London Olympics last summer. How, at a time when Webb was theoretically entering his physical peak, did he fall from breaking 3:47 in the mile in 2007 to barely breaking 4 minutes in 2012?

“What has changed the most has been physiological,” Webb says. “I have to factor in that I have gotten older. My lack of success has been in me trying to adapt to that.” He describes the olden days — his late teens and early 20s — as ones where he could “do anything I wanted [physically in hard training] and it always worked out for me. Even when things went wrong I always bounced back.”

Specifically, Webb cites two other factors that have hindered his performances. In 2008, he says he hit the weight room too hard. Not crazy stuff like maxing out on the bench press, but routines where he put 150 pounds on the bar and did three sets of 15. It was what he did in high school — hell, it was what every miler in America learned in high school: the football players grunt and grind and lift big for huge muscles, while the long-distance runners do lots of lightweight reps. Except, notes Webb, he started to bulk up on that iron diet and developed the most talked about biceps in long-distance running.

RELATED: Adding Relative Strength

“Everyone says do more reps, so I did that, but I wanted it to be hard so I did it all hard,” Webb says. “But you blow up.” Instead of making running easier, lifting did the opposite. Although he was still doing the same kind of running, he’d put on five pounds and didn’t have the same fluidity in his form or snap in his legs.

Today, after adapting to a new vegetable-friendly nutrition plan and additional weekly mileage, Webb is back down to 140 pounds, the lightest he’s been in several years.

“I did some bench press in high school, but I’d win races anyways,” Webb says matter-of-factly. “At the professional level, you put on five pounds and you go from setting the American record to the back.”

To illustrate the change between then and now, he uses a thoughtful shooting analogy. As he explains, the first half of his career felt like he was aiming at a target 10 yards away with a shotgun. The few times he wasn’t perfectly on target, some of the pellets still easily hit the bull’s eye. Lately, though, he feels like he’s using a Civil War musket to try and hit a target 100 meters away while aiming blindfolded. “My margin of error is so much smaller,” assesses Webb, who turned 30 earlier this year.

Webb admits he now wonders if he should have focused on the 5K and 10K more seriously the year after he set the American mile record. When he ran his 5000m PR of 13:10.86 in 2005, he was the third-fastest U.S. runner of all-time, but Tegenkamp, Solinsky, Dathan Ritzenhein and Galen Rupp have all passed him by and joined American record-holder Lagat in the sub-13:00 club. Still, Webb realizes, he had unfinished business in the four-lap race. “I had the dream to be the best miler in the world and held onto it,” he says. “It was hard to give up.”

Instead of moving up in distance, he moved from one coach to the next every few years. They were some of the brightest minds in track and field: Alberto Salazar, Jason Vigilante and Scott Raczko, the man who guided Webb to both his prep and professional mile records. Webb never stayed long at each stop because he could never achieve the one thing he was seeking — perfection, the unattainable pedestal of beating the best in the world every time he faced them. Now, as he looks back, he realizes why he never found perfection. “I started to realize it’s not there,” he says. “It doesn’t exist.”

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