racl2 ( bayer healthcare pharmaceuticals , leverkusen , germany ) was calibrated according to guidelines provided by the supplier in response to the nuclear regulator commission ( 14 ) . briefly , the dial setting of the calibrator ( crc-127r , capintec ; ramsey , new jersey ) was determined by selecting a value that was confirmed with the decay - corrected national institute of standards and technology standard calibrated dose upon vialing . animals received 150 l of clinical - grade racl2 citrate solution , diluted in sterile physiological saline , immediately before injection . cd-1 mice ( charles river laboratories ) , b6.cg c57bl/6 mice ( jackson laboratories ; bar harbor , me ) , and nu / nu mice ( harlan laboratories ; indianapolis , in ) were placed on standard feed ad libitum . all mouse experimentation was in accordance with institutional animal welfare protocols at the johns hopkins university school of medicine . pc3 and lncap - ar cells ( 15 ) were inoculated intratibially in nu / nu mice , as described ( 16 ) . bone metastasis was monitored by bioluminescence and x - ray computed tomography ( ct ) using the ivis spectrumct ( perkin elmer ; waltham , ma ) . mice were dosed with racl2 ( 10 kbq , n = 46 per experimentation group ) and killed at one , four , or 24 hours for prompt dissection . to determine remnant whole - body activity at these time points , tissue activity was assayed by gamma (  ) counting or was cryo - embedded ( oct , sakura fintec , torrance , ca ) . for whole - body autoradiography samples , no fixation or decalcification chemicals were applied to mouse tissues , which were either stored ( -80 c ) or flash - frozen in liquid nitrogen . the main photopeak detected at 270 kev was provided by ra ( 269 kev ; 13.6% ) and rn ( 271 kev ; 9.9% ) emissions ( wizard , perkin elmer ) . high - resolution ct ( hrct ) , histological and autoradiographic methods are described in the supplementary materials ( available online ) . statistical evaluation was performed in prism 5.0d ( graphpad software ; la jolla , ca ) and excel ( microsoft ; redmond , wa ) using an unpaired , two - tailed student s t test . differences at the 95% confidence level ( p < .05 ) were considered to be statistically significant . racl2 ( bayer healthcare pharmaceuticals , leverkusen , germany ) was calibrated according to guidelines provided by the supplier in response to the nuclear regulator commission ( 14 ) . briefly , the dial setting of the calibrator ( crc-127r , capintec ; ramsey , new jersey ) was determined by selecting a value that was confirmed with the decay - corrected national institute of standards and technology standard calibrated dose upon vialing . animals received 150 l of clinical - grade racl2 citrate solution , diluted in sterile physiological saline , immediately before injection . cd-1 mice ( charles river laboratories ) , b6.cg c57bl/6 mice ( jackson laboratories ; bar harbor , me ) , and nu / nu mice ( harlan laboratories ; indianapolis , in ) were placed on standard feed ad libitum . all mouse experimentation was in accordance with institutional animal welfare protocols at the johns hopkins university school of medicine . pc3 and lncap - ar cells ( 15 ) were inoculated intratibially in nu / nu mice , as described ( 16 ) . bone metastasis was monitored by bioluminescence and x - ray computed tomography ( ct ) using the ivis spectrumct ( perkin elmer ; waltham , ma ) . mice were dosed with racl2 ( 10 kbq , n = 46 per experimentation group ) and killed at one , four , or 24 hours for prompt dissection . to determine remnant whole - body activity at these time points , tissue activity was assayed by gamma (  ) counting or was cryo - embedded ( oct , sakura fintec , torrance , ca ) . for whole - body autoradiography samples , mice were killed at 24 hours ( n = 3 ) . no fixation or decalcification chemicals were applied to mouse tissues , which were either stored ( -80 c ) or flash - frozen in liquid nitrogen . the main photopeak detected at 270 kev was provided by ra ( 269 kev ; 13.6% ) and rn ( 271 kev ; 9.9% ) emissions ( wizard , perkin elmer ) . high - resolution ct ( hrct ) , histological and autoradiographic methods are described in the supplementary materials ( available online ) . statistical evaluation was performed in prism 5.0d ( graphpad software ; la jolla , ca ) and excel ( microsoft ; redmond , wa ) using an unpaired , two - tailed student s t test . differences at the 95% confidence level ( p < .05 ) were considered to be statistically significant . previous studies in rodents have found minimal ( 17 ) or undetectable ( 18 ) uptake of ra outside of the calcified tissues . this contrasts with the human planar imaging of therapeutic doses of the radiopharmaceutical , which demonstrated intense abdominal uptake ( 13 ) . to test the utility of mice as models localization to the bone was rapid , with 1.85%  0.49% , 2.86%  0.49% , and 4.54%  0.65% injected activity ( ia ) in the tibia , femur , and vertebrae samples after one hour determined by -counting ( figure 2a ) . bone labeling at four and 24 hours was non - statistically significant from the initial reading , and this confirms previous reports that radium-223 ( and its short - lived daughters ) do not redistribute in rodent or man ( 13,17 ) . we therefore focused our interest on the acute distribution of the radionuclide . adjusting for the mass of resected tissues for the readings , at 24 hours postadministration we measured 51.19%  11.53% , 46.68%  5.12% , and 30.78% 3.99% ia per gram ( % ia / g ) in the tibia , femur , and vertebrae , respectively ( figure 2b ) . mice ( n = 45 per group ) were killed at one , four , and 24 hours postinjection , and organs evaluated for activity by  counting . a ) the distribution is expressed in percent injected activity ( % ia  sd ) representing the amount in each organ relative to the administered dose and ( b ) normalized to the weight of each organ ( % ia / g  standard error of the mean ) . c ) ra clearance profile was defined measuring whole - body radioactive content over 24 hours . d - f ) to confirm ex vivo  count results , whole - body undecalcified cryosections ( 14 m ) were obtained at 24 hours after administration . autoradiography revealed skeletal uptake in the calvaria and vertebrae , while soft tissue accumulation is recapitulated in the spleen and kidney . uptake in the spleen , stomach , and intestinal organs and the expected renal clearance and early bladder passage ( genitourinary package ) were observed ( figure 2 ) . these results underline the non - negligible levels of ra outside of the bone and reveal that a statistically significant fraction ( > 25% ia ) of the agent rapidly transits to the small and large intestine to be excreted ( figure 2a ) . ra uptake in the bones ( > 30% injected activity per gram ) at 24 hours was also accompanied by non - negligible remnant activity in the kidney ( 2.33%  0.36% ) , intestines ( 5.73%  2.04% ) , and spleen ( 10.5%  5.9% ) ( figure 2b ) . combined renal and intestinal clearance resulted in retention of 81%  5% of the ia at one hour , which decreased at four hours to 76%  3% and fell further at 24 hours to only 46.5%  2% . these values resemble those determined in a phase i pharmacokinetic study in men with bmcrpc ( 13 ) and validate the use of skeletally mature rodents as a model to study radium-223 in men . to verify  counts , whole - body autoradiography visually confirmed soft - tissue and skeletal uptake . standard tissue processing for histochemistry and evaluation ( including fixation and decalcification ) would remove ra or alter the radionuclide distribution . thus , mice were killed and flash frozen at 24 hours postinjection , prior to unfixed and undecalcifed sectioning . macrographs , autoradiography , and overlay are shown for representative sections ( figure 2 , d - f ) . direct imaging corroborates  counts with prominent ra labeling of the skeleton , spleen , stomach , and kidneys . autoradiography and  counting revealed non - negligible soft - tissue uptake ( figure 2 ) . c57bl/6 mice are known to be osteopenic relative to other strains ( 19 ) , and it is conceivable that use of this low - bone model may bias ra results towards lower bone uptake and greater intestinal accumulation . to interrogate the role of bone phenotype on uptake , we evaluated distribution in a second common strain . skeletally mature , age - matched , male c57bl/6 and cd-1 mice were dosed with racl2 , and distribution was evaluated . uptake was not statistically significant between the strains for any of the soft tissues ( figure 3 , a and b ) . the minimum statistical test value across the soft tissues was the small intestine ( % ia / g , p = .39 ) . however , greater labeling in the c57bl/6 strain was noted in the skeleton , in total counts , when compared with cd-1 uptake ( figure 3a ) . these differences were statistically significant for the femur ( p < .001 ) and tibia ( p = .04 ) and trended in that direction for the vertebrae . the distinction between the two strains was magnified when adjusting for bone mass ( % ia / g ) ( figure 3b ) as c57bl/6 have reduced skeletal volume . indeed , c57bl/6 mice ( 14.720.11 mm ) have grossly smaller femur length than that of the cd-1 strain ( 15.620.26 mm , p = .04 ) ( figure 3b insert ) . a ) the distribution of the radionuclide was compared in age- and sex - matched c57bl/6 and cd-1 ( n = 5 ) as percent injected activity % ia  sd . b ) the greater overall uptake in the smaller long bones of the c57bl/6 strain ( insert ) is enhanced when normalized to tissue mass ( % ia / g  sd ) . three - dimensional bone volume from representative samples of ( d ) the trabecular ( at two perspectives ) and ( f ) cortical regions of the femur ( scale bars = 500 m ) . e ) quantitative ct analysis of the distal femur reveals the larger tissue and bone volume of the cd-1 mice . the bone density bone volume fraction ( bv / tv  standard error of the mean ) and other morphologic parameters of the trabeculae do not meet statistical significance ; therefore , there is incontrovertibly greater bone area in the cd-1 mice . g ) likewise , analysis of the cortical bone establishes the greater size of the cd-1 ( tissue and bone area  sd ) to that of c57bl/6 , despite the two strains having similar cortical area fraction values ( bone / tissue area  sd ) . statistical significance was determined using an unpaired two - tailed student s t test ; * p < dependent features of bone microarchitecture might explain the difference in ra distribution . to ask this question , we undertook detailed hrct of the trabecular and cortical regions of the femur and tibia ( femur schematic ) ( figure 3c ) . the bone volume of the cd-1 was greater than that of the c57bl/6 in the trabecular and cortical regions of the femur ( figure 3 , d and f ) . quantitative analysis confirmed that the cd-1 strain exhibits greater total tissue volume ( 5.310.95 mm vs 3.560.20 mm ; p = .02 ) and occupying bone volume ( 1.0810.18 mm vs 0.7810.11 mm ; p = .04 ) in the cancellous bone than c57bl/6 . however , there was no difference in the density or thickness of trabecular structures ( p = .21 ) . consequently , bone as a percent of the total tissue volume ( bv / tv ) was not statistically significantly different , at approximately 20% ( p = .35 ) ( figure 3e ) . the amount of total tissue in the cd-1 was statistically significantly greater than the c57bl/6 ( 2.330.137 mm compared with 1.730.077 mm ; p = .02 ) while the cortical area fraction , or bone - to - tissue area ratio , does not vary statistically significantly , at nearly 45% ( p = .09 ) ( figure 3 g ) . therefore , the paradoxically greater uptake in smaller bones could not be explained by a bone tissue area balance at the micron scale ; cd-1 mice have statistically significantly more total bone and bone surface area yet lower ra uptake . racl2 was rapidly cleared from the blood ( figure 2 , a and b ) , and we next tested the hypothesis that discrepant skeletal uptake could be attributed to differences in vascular delivery . the number of vessels in the trabecular compartment was computed by counting the number of cd31-positive vessels in 40 high - powered fields in the tibia and femur . the c57bl/6 samples had a greater vascular density of cd31-positive vessels than that of cd-1 ( figure 4b ) . there was an approximately 28%  8% increase in the number of blood vessels per mm across age- and sex - matched mice from the two strains ( p = .04 ) . no statistically significant difference was found in the marrow cavity ( data not shown ) . these results suggest that a dominant characteristic for the magnitude of ra uptake at bone sites is blood vessel density dependent rather than the quantity of exposed bone surface area or bone volume . a ) vessels identified in the c57bl/6 and cd-1 strains of mice ( n = 3 per strain ) in the trabecular and cortical compartments , indicated by arrowhead ( scale bar = 20 m ) . error bars represent sd , and statistical significance was determined using an unpaired two - tailed student s t test ; * p < .05 . intact hind limbs of nave mice were cryosectioned 24 hours following dosing by racl2 and scanned by  camera . a representative section is shown , including : en bloc macrograph of the sample cutting surface on the cryostat , a white light reflectance image of the tissue section in the  camera ,  exposure , and overlay ( figure 5 , a - d ) . the system resolution ( ~50 m ; measured using a optical line target ) revealed localization predominantly to the distal femur and proximal tibia , with little activity in the marrow cavity or cortical bone surface . safranin - o differentiates mineral bone ( in green ) from proteoglycans and cartilage ( in red - orange ) . binding to the cartilage itself is not observed , either at the growth plate or at the articular surfaces of the joints ( figure 5e ) . ra distribution colocalized with the freshly mineralized osteoid matrix characterized by a very high bone modeling / remodeling activity directly at the front of the growth plate . undecalcified whole - mount cryosection and alpha camera autoradiography of distal femur and proximal tibia . b ) white - light reflectance image of alpha - scintillant mounted section ( 14 m ) in  camera . c )  camera acquisition of radium-223 ( and -emitting daughters ) distribution in sample . e ) composite microscope image of safranin - o histology revealing mature bone ( green ) and uncalcified proteoglycans ( orange ) at the growth plate , adjacent to the ossification front . representative result of n = 6 ; scale bars = 500 m . using an intratibial inoculation model of metastasis , we evaluated the acute microdistribution of ra with osteoblastic ( lncap ) and osteolytic ( pc3 ) pca lines . en bloc macrophotographs and cryosections were exposed and developed histochemically ( hematoxylin and eosin , safranin - o ) for tissue morphology identification ( figure 6 ) . representative autoradiograms demonstrate that ra does not localize directly to the tumor ( indicated by arrow ) , regardless of type . instead , activity accumulates at the apposite bone surface surrounding the lesion , as well as again at the active bone modeling / remodeling sites . the ra deposition in healthy bone sites is likewise equivalent to the levels in bone surrounding the tumors . further , alkaline phosphatase enzyme stain ( alp ) colocalized osteoblastic activity and ra uptake at both the malignant and joint site ( supplementary figure 1 , available online ) . undecalcified whole - mount cryosection and autoradiography of osteoblastic ( lncap ) and osteolytic ( pc3 ) intratibial prostate cancer xenograft models ( n = 34 mice per group ) . haematoxylin and eosin ( h&e ) stain of the whole - mount adhesive bound section , tumor indicated by arrow . safranin - o staining for calcified ( green ) and uncalcified ( orange - brown ) regions . areas of intense uptake colocalize with active bone modeling / remodeling sites ( at the interface of the calcified and uncalcified compartments ) in the distal femur , proximal tibia , and the bone surfaces adjacent to the bone metastasis . previous studies in rodents have found minimal ( 17 ) or undetectable ( 18 ) uptake of ra outside of the calcified tissues . this contrasts with the human planar imaging of therapeutic doses of the radiopharmaceutical , which demonstrated intense abdominal uptake ( 13 ) . to test the utility of mice as models localization to the bone was rapid , with 1.85%  0.49% , 2.86%  0.49% , and 4.54%  0.65% injected activity ( ia ) in the tibia , femur , and vertebrae samples after one hour determined by -counting ( figure 2a ) . bone labeling at four and 24 hours was non - statistically significant from the initial reading , and this confirms previous reports that radium-223 ( and its short - lived daughters ) do not redistribute in rodent or man ( 13,17 ) . we therefore focused our interest on the acute distribution of the radionuclide . adjusting for the mass of resected tissues for the readings , at 24 hours postadministration we measured 51.19%  11.53% , 46.68%  5.12% , and 30.78% 3.99% ia per gram ( % ia / g ) in the tibia , femur , and vertebrae , respectively ( figure 2b ) . mice ( n = 45 per group ) were killed at one , four , and 24 hours postinjection , and organs evaluated for activity by  counting . a ) the distribution is expressed in percent injected activity ( % ia  sd ) representing the amount in each organ relative to the administered dose and ( b ) normalized to the weight of each organ ( % ia / g  standard error of the mean ) . c ) ra clearance profile was defined measuring whole - body radioactive content over 24 hours . d - f ) to confirm ex vivo  count results , whole - body undecalcified cryosections ( 14 m ) were obtained at 24 hours after administration . autoradiography revealed skeletal uptake in the calvaria and vertebrae , while soft tissue accumulation is recapitulated in the spleen and kidney . uptake in the spleen , stomach , and intestinal organs and the expected renal clearance and early bladder passage ( genitourinary package ) were observed ( figure 2 ) . these results underline the non - negligible levels of ra outside of the bone and reveal that a statistically significant fraction ( > 25% ia ) of the agent rapidly transits to the small and large intestine to be excreted ( figure 2a ) . ra uptake in the bones ( > 30% injected activity per gram ) at 24 hours was also accompanied by non - negligible remnant activity in the kidney ( 2.33%  0.36% ) , intestines ( 5.73%  2.04% ) , and spleen ( 10.5%  5.9% ) ( figure 2b ) . combined renal and intestinal clearance resulted in retention of 81%  5% of the ia at one hour , which decreased at four hours to 76%  3% and fell further at 24 hours to only 46.5%  2% . these values resemble those determined in a phase i pharmacokinetic study in men with bmcrpc ( 13 ) and validate the use of skeletally mature rodents as a model to study radium-223 in men . to verify  counts , whole - body autoradiography visually confirmed soft - tissue and skeletal uptake . standard tissue processing for histochemistry and evaluation ( including fixation and decalcification ) would remove ra or alter the radionuclide distribution . thus , mice were killed and flash frozen at 24 hours postinjection , prior to unfixed and undecalcifed sectioning . macrographs , autoradiography , and overlay are shown for representative sections ( figure 2 , d - f ) . direct imaging corroborates  counts with prominent ra labeling of the skeleton , spleen , stomach , and kidneys . autoradiography and  counting revealed non - negligible soft - tissue uptake ( figure 2 ) . c57bl/6 mice are known to be osteopenic relative to other strains ( 19 ) , and it is conceivable that use of this low - bone model may bias ra results towards lower bone uptake and greater intestinal accumulation . to interrogate the role of bone phenotype on uptake , we evaluated distribution in a second common strain . skeletally mature , age - matched , male c57bl/6 and cd-1 mice were dosed with racl2 , and distribution was evaluated . uptake was not statistically significant between the strains for any of the soft tissues ( figure 3 , a and b ) . the minimum statistical test value across the soft tissues was the small intestine ( % ia / g , p = .39 ) . however , greater labeling in the c57bl/6 strain was noted in the skeleton , in total counts , when compared with cd-1 uptake ( figure 3a ) . these differences were statistically significant for the femur ( p < .001 ) and tibia ( p = .04 ) and trended in that direction for the vertebrae . the distinction between the two strains was magnified when adjusting for bone mass ( % ia / g ) ( figure 3b ) as c57bl/6 have reduced skeletal volume . indeed , c57bl/6 mice ( 14.720.11 mm ) have grossly smaller femur length than that of the cd-1 strain ( 15.620.26 mm , p = .04 ) ( figure 3b insert ) . a ) the distribution of the radionuclide was compared in age- and sex - matched c57bl/6 and cd-1 ( n = 5 ) as percent injected activity % ia  sd . b ) the greater overall uptake in the smaller long bones of the c57bl/6 strain ( insert ) is enhanced when normalized to tissue mass ( % ia / g  sd ) . three - dimensional bone volume from representative samples of ( d ) the trabecular ( at two perspectives ) and ( f ) cortical regions of the femur ( scale bars = 500 m ) . e ) quantitative ct analysis of the distal femur reveals the larger tissue and bone volume of the cd-1 mice . the bone density bone volume fraction ( bv / tv  standard error of the mean ) and other morphologic parameters of the trabeculae do not meet statistical significance ; therefore , there is incontrovertibly greater bone area in the cd-1 mice . g ) likewise , analysis of the cortical bone establishes the greater size of the cd-1 ( tissue and bone area  sd ) to that of c57bl/6 , despite the two strains having similar cortical area fraction values ( bone / tissue area  sd ) . statistical significance was determined using an unpaired two - tailed student s t test ; * p < dependent features of bone microarchitecture might explain the difference in ra distribution . to ask this question , we undertook detailed hrct of the trabecular and cortical regions of the femur and tibia ( femur schematic ) ( figure 3c ) . the bone volume of the cd-1 was greater than that of the c57bl/6 in the trabecular and cortical regions of the femur ( figure 3 , d and f ) . quantitative analysis confirmed that the cd-1 strain exhibits greater total tissue volume ( 5.310.95 mm vs 3.560.20 mm ; p = .02 ) and occupying bone volume ( 1.0810.18 mm vs 0.7810.11 mm ; p = .04 ) in the cancellous bone than c57bl/6 . however , there was no difference in the density or thickness of trabecular structures ( p = .21 ) . consequently , bone as a percent of the total tissue volume ( bv / tv ) was not statistically significantly different , at approximately 20% ( p = .35 ) ( figure 3e ) . the amount of total tissue in the cd-1 was statistically significantly greater than the c57bl/6 ( 2.330.137 mm compared with 1.730.077 mm ; p = .02 ) while the cortical area fraction , or bone - to - tissue area ratio , does not vary statistically significantly , at nearly 45% ( p = .09 ) ( figure 3 g ) . therefore , the paradoxically greater uptake in smaller bones could not be explained by a bone tissue area balance at the micron scale ; cd-1 mice have statistically significantly more total bone and bone surface area yet lower ra uptake . racl2 was rapidly cleared from the blood ( figure 2 , a and b ) , and we next tested the hypothesis that discrepant skeletal uptake could be attributed to differences in vascular delivery . the number of vessels in the trabecular compartment was computed by counting the number of cd31-positive vessels in 40 high - powered fields in the tibia and femur . the c57bl/6 samples had a greater vascular density of cd31-positive vessels than that of cd-1 ( figure 4b ) . there was an approximately 28%  8% increase in the number of blood vessels per mm across age- and sex - matched mice from the two strains ( p = .04 ) . no statistically significant difference was found in the marrow cavity ( data not shown ) . these results suggest that a dominant characteristic for the magnitude of ra uptake at bone sites is blood vessel density dependent rather than the quantity of exposed bone surface area or bone volume . a ) vessels identified in the c57bl/6 and cd-1 strains of mice ( n = 3 per strain ) in the trabecular and cortical compartments , indicated by arrowhead ( scale bar = 20 m ) . error bars represent sd , and statistical significance was determined using an unpaired two - tailed student s t test ; * p < .05 . intact hind limbs of nave mice were cryosectioned 24 hours following dosing by racl2 and scanned by  camera . a representative section is shown , including : en bloc macrograph of the sample cutting surface on the cryostat , a white light reflectance image of the tissue section in the  camera ,  exposure , and overlay ( figure 5 , a - d ) . the system resolution ( ~50 m ; measured using a optical line target ) revealed localization predominantly to the distal femur and proximal tibia , with little activity in the marrow cavity or cortical bone surface . safranin - o differentiates mineral bone ( in green ) from proteoglycans and cartilage ( in red - orange ) . binding to the cartilage itself is not observed , either at the growth plate or at the articular surfaces of the joints ( figure 5e ) . ra distribution colocalized with the freshly mineralized osteoid matrix characterized by a very high bone modeling / remodeling activity directly at the front of the growth plate . undecalcified whole - mount cryosection and alpha camera autoradiography of distal femur and proximal tibia . b ) white - light reflectance image of alpha - scintillant mounted section ( 14 m ) in  camera . c )  camera acquisition of radium-223 ( and -emitting daughters ) distribution in sample . e ) composite microscope image of safranin - o histology revealing mature bone ( green ) and uncalcified proteoglycans ( orange ) at the growth plate , adjacent to the ossification front . using an intratibial inoculation model of metastasis , we evaluated the acute microdistribution of ra with osteoblastic ( lncap ) and osteolytic ( pc3 ) pca lines . en bloc macrophotographs and cryosections were exposed and developed histochemically ( hematoxylin and eosin , safranin - o ) for tissue morphology identification ( figure 6 ) . representative autoradiograms demonstrate that ra does not localize directly to the tumor ( indicated by arrow ) , regardless of type . instead , activity accumulates at the apposite bone surface surrounding the lesion , as well as again at the active bone modeling / remodeling sites . the ra deposition in healthy bone sites is likewise equivalent to the levels in bone surrounding the tumors . further , alkaline phosphatase enzyme stain ( alp ) colocalized osteoblastic activity and ra uptake at both the malignant and joint site ( supplementary figure 1 , available online ) . undecalcified whole - mount cryosection and autoradiography of osteoblastic ( lncap ) and osteolytic ( pc3 ) intratibial prostate cancer xenograft models ( n = 34 mice per group ) . haematoxylin and eosin ( h&e ) stain of the whole - mount adhesive bound section , tumor indicated by arrow . safranin - o staining for calcified ( green ) and uncalcified ( orange - brown ) regions . areas of intense uptake colocalize with active bone modeling / remodeling sites ( at the interface of the calcified and uncalcified compartments ) in the distal femur , proximal tibia , and the bone surfaces adjacent to the bone metastasis . metastatic invasion of the skeleton in pca results in pain , risk of fracture , and bone marrow failure . rodent models have provided considerable value in development of bmcrpc drug candidates ( 15,20 ) . however , there is scant literature evaluating racl2 in preclinical models of pca , with notable exceptions ( 18 ) . our investigation focused on understanding the pharmacodynamics of ra and the development of tools to enable its detailed study at multiple scales . whole - body ra distribution was determined and compared with bone features including hrct and vasculature morphology . the skeletally mature mice ( age > 14 weeks ) used in this work possess normalized bone growth and resorption rates ( 21 ) . the epiphyseal plates of mice do not fuse , and ra accumulation was noted at sites of bone activity  whether adjacent to the growth plate or surrounding implanted intraosseous xenografts . along with expected skeletal accumulation , non - negligible uptake in the spleen , intestines , and kidney was quantified and imaged and corresponded with the gastrointestinal accumulation seen in men using planar -scintigraphy ( 13,22 ) . clinical effects of uptake at these sites are associated with reported adverse gastrointestinal events , often leading to cessation of treatment ( 23 ) . acknowledging differences between mouse and human bone properties , these results provide support for the use of skeletally mature mouse models as surrogates to evaluate the impact of radium-223 ( 24 ) . statistically significantly greater ra % ia ( p < .04 ) and % ia / g ( p < .001 ) in the tibia and femur of smaller c57bl/6 over cd-1 mice was observed . c57bl/6 mice possess less bone volume and area but similar bone density and trabecular number , ruling out a morphometric explanation for the unexpected result . ra has a short circulation time , and enhanced uptake may be the result of the greater c57bl/6 microvascular density determined by anti - cd31 staining . there was no statistically significant difference in uptake in the vertebrae ; however , differences in the appendicular and axial skeleton , as well as the difficulty in fully removing soft - tissue from the spine , may explain this result . the abundance - weighted average distance of  emissions from the ra decay chain is 57 m ( rtissue ) , leading us to focus on microstructural localization of the agent . previous reports of suborgan distribution are limited to investigation of ra isotopes by solution autoradiography in fixed , methyl - methacrylate - embedded specimens ( 25,26 ) or scanning photometry ( 27 ) , not readily available techniques . using autoradiography and advancing a recently described  camera system ( 28 ) , ra distribution was imaged at physiological doses . in the metabolically active sites of the mouse skeleton , such as the distal femur and proximal tibia , ra localized to the ossification sites contiguous to the growth plate ( figure 5 ) . imaging ra directly is difficult in patients , and questions remain regarding uptake across patients and between lesions . an intratibial inoculation model was used to evaluate the acute microdistribution in models of pca bone metastasis with both an osteoblastic ( lncap ) and osteolytic ( pc-3 ) phenotype . at bone adjacent to the intraosseous tumors , bone remodeling was confirmed by safarinin - o and alp ( supplemental figure 1 , available online ) and colocalized with ra - labeling ( figure 6 ) . uptake in the bone microenvironment surrounding metastases occurred at a similar intensity for both osteoblastic and osteolytic tumors . this may have important implications as -therapy is applied to other osteotropic malignancies including breast , renal , lung , and thyroid carcinoma . localization to malignant sites was similar in intensity to the background activity at the epiphyses . this presents a limitation for use of mice as a surrogate for man in studying the whole - body effects of ra but may enable study of radiobiological effects at sites of non - tumor - involved bone activity ( such as at degenerative skeletal foci found throughout the geriatric cancer population ) . from the preclinical perspective , these results underline that antineoplastic effect may be dependent on tumor model parameters including metastasis volume , distance to vascularized bone surfaces , and tumor placement . models using lesions directly adjacent to epiphyseal plate will be irradiated by ra , which may complicate evaluation ( 29 ) . to match the clinical indication , we examined ra localization to bone lesions using intraosseous inoculation ( exempt of visceral disease that accompanies systemic pca cell injection ) . other bone metastatic models or techniques may be useful to evaluate alternate contexts such as the degree of localization to incipient lesions ( prior to fracture ) or in the axial skeleton . long - term treatment studies in these mouse models are currently ongoing . our results here suggest that increases in activity will contribute dose around the metastasis rather that throughout the lesion , that resorptive lesions may be valid targets for ra , and that compromising vascular access to bone lesions may reduce ra delivery . ongoing clinical trials using ra in combination with antiangiogenic tyrosine kinase inhibitors or tasquinimod may shed more light on the connection between treatment efficacy and osteovasculature ( nct02406521 and nct02406521 ) . we investigated the features of ra distribution in healthy and diseased mice to establish their use towards improved application of this clinical bone - seeking  emitter . quantitative biodistribution revealed an uptake profile closely matching man at the organ scale . utilizing microstructural , histological , and emerging nuclear imaging tools , we have established suborgan ra deposition to areas of metabolically active bone with a dependence on local blood vessel density . our results encourage further use of advanced preclinical models and the techniques developed here to guide combination treatment paradigms and application to other malignancies . this work was supported by the prostate cancer foundation through the steve wynn young investigator award ( dljt ) , the david h. kock young investigator award ( du ) , the patrick c. walsh fund ( dljt ) , and the society of nuclear medicine and molecular imaging junior faculty fund ( dsa ) . we acknowledge national institutes of health funding for the prostate spore p50ca058236 and r01 ca157542 ( dsa , rfh ) .
background : bone - metastatic , castration - resistant prostate cancer ( bmcrpc ) represents a lethal stage of the most common noncutaneous cancer in men . 
 the recent introduction of radium-223 dichloride , a bone - seeking alpha particle ( )emitting radiopharmaceutical , demonstrates statistically significant survival benefit and palliative effect for bmcrpc patients . 
 clinical results have established safety and efficacy , yet questions remain regarding pharmacodynamics and dosing for optimized patient benefit.methods:we elucidated the biodistribution of 223ra as well as interaction with the bone and tumor compartments in skeletally mature mice ( c57bl/6 and cd-1 , n = 36 ) and metastasis models ( lncap and pc3 , n = 4 ) . 
 differences in uptake were evaluated by ct and histological investigation . 
 novel techniques were leveraged on whole - mount undecalcified cryosections to determine microdistribution of radium-223 . 
 all statistical tests were two-sided.results : 
 223ra uptake in the bones ( > 30% injected activity per gram ) at 24 hours was also accompanied by non - negligible remnant activity in the kidney ( 2.33%  0.36% ) , intestines ( 5.73%  2.04% ) , and spleen ( 10.5%  5.9% ) skeletal accumulation across strains did not correspond with bone volume or surface area but instead to local blood vessel density ( p = .04 ) . 
 microdistribution analysis by autoradiography and  camera revealed targeting of the ossifying surfaces adjacent to the epiphyseal growth plate . in models of pca metastasis , radioactivity does not localize directly within tumors but instead at the apposite bone surface . 
 osteoblastic and lytic lesions display similar intensity , which is comparable with uptake at sites of normal bone remodeling.conclusions:profiling the macro- and microdistribution of 223ra in healthy and diseased models has important implications to guide precision application of this emerging -therapy approach for bmcrpc and other bone metastastic diseases .