following a clinically predictable progression 
 pattern , advanced 
 prostate cancer ( pca ) metastasizes to distant organs , with a striking 
 predisposition for bone . despite a decreasing 
 overall mortality rate for pca patients ,
the development 
 of effective treatments has been hindered , in part , by the lack of 
 cell lines and/or xenograft models that accurately recapitulate the 
 complex metastatic microenvironment . without appropriate 
 models to reflect the disease , mechanistic studies to accurately elucidate 
 the players involved in pca progression in bone have been difficult 
 to implement , impeding the development of clinically effective therapeutics 
 targeted to bone metastases .
some 
 of the more commonly used pca cell lines ( e.g. , pc-3 and du-145 ) 
 offer convenience to investigators because they adhere well to tissue 
 culture plastic and therefore are amenable to high throughput screening 
 of drug libraries .
however , these same cell lines , when inoculated 
 in bone , generate a largely osteolytic response , in contrast to the 
 predominantly osteoblastic nature of the native human disease .
to 
 more accurately model the disease , we have employed the lncap cell 
 line progression series ( lncap , c4 , c4 - 2 , and c4 - 2b ) , with c4 - 2b cells 
 forming osseous lesions in bone .
however , 
 we and other investigators continue to search for pca cell models 
 with greater fidelity to the disease that will foster the translation 
 of preclinical findings into the clinic , particularly to satisfy the 
 need to identify new treatments that will eradicate pca metastases 
 growing in bone . to address the need for the highest - possible 
 fidelity in pca cell 
 sources , patient - derived xenograft ( pdx ) models
have been established 
 for the preclinical investigation of various aspects of pca biology 
 including angiogenesis , identification of castrate - resistant stem - like 
 cells , and effect of anti - androgen therapies .
pdx models are generated when 
 tumor tissue from the patient is surgically resected and engrafted 
 directly into immunocompromised mice .
tumors 
 are subsequently maintained solely in vivo via mouse - to - mouse passage , 
 requiring both careful monitoring of the tumor burden and labor intensive 
 animal transfers . through serial passaging 
 in mice and the absence of any in vitro manipulation ,
pdx tumors remain 
 biologically stable , preserving much of the molecular , genetic , and 
 histological features as well as heterogeneity of the original tumor .
however , given the high costs of animal maintenance , 
 lengthy latency period following engraftment , variable engraftment 
 rates , and rare access to patient tissue specimens , pdx in vivo models 
 are generally not yet widely employed in cancer research .
most notably , given the poor viability exhibited 
 when grown in vitro under standard tissue culture conditions , it is 
 extremely challenging to culture pdx bone metastatic pca cells for 
 any brief ex vivo manipulation needed to conduct controlled mechanistic 
 studies in vivo .
mirroring the in vitro behavior of pdx prostate 
 tumors , pca cell 
 lines belonging to the lncap series also adhere poorly to two - dimensional 
 ( 2d ) tissue culture surfaces , precluding them from use in standard 
 drug screening platforms . to circumvent 
 this problem ,
our laboratory recently demonstrated the feasibility 
 of using three - dimensional ( 3d ) hyaluronan ( ha)-based hydrogel systems 
 to support the growth and viability of these pca cell lines for mechanistic 
 studies and drug testing . while the commonly employed human 
 tumor spheroid model is an alternative to culturing these poorly adherent 
 cells , hydrogel encapsulation provides the means to fully recapitulate 
 the tumor microenvironment with precise , tunable control over architectural 
 and mechanical cues and/or critical cell 
specifically , as a ubiquitous component of the bone marrow 
 where bone metastatic pca cells reside , ha plays an active role in 
 regulating several biological processes , including tumorigenesis , 
 strongly justifying its use as an extracellular matrix analogue for 
 culturing bone metastatic tumor cells in vitro . on the basis of the suitability of ha - based hydrogel 
 systems for 
 the culture of pca cell lines , we hypothesized that these systems 
 would similarly support the viability of pdx pca cells in vitro .
hence , 
 in the present study , we developed a novel protocol to encapsulate 
 pdx pca cells within 3d ha - based hydrogels and examined tumor cell 
 morphology , viability , proliferative capacity and phenotype .
we also 
 tested the potential of this 3d pdx pca model as a diagnostic platform 
 for evaluating rapid patient - specific drug response , a significant 
 advance toward achieving a more personalized therapeutic regimen .
phenol red - free dulbecco s modified 
 eagle medium / nutrient mixture f-12 ( dmem / f-12 ) , t - medium , penicillin / streptomycin , 
 trypsin - edta , l - glutamine , live / dead viability / cytotoxicity 
 kit , and quant - it picogreen dsdna assay kit were obtained from life 
 technologies ( grand island , ny ) . fetal bovine serum ( fbs )
phosphate buffered 
 saline ( pbs ) was obtained from lonza ( walkersville , md ) .
thiol - modified ha ( ha - sh , 
 glycosil , average mw = 240 kda , degree 
 of thiolation = 1 mol / mg ha - sh ) and poly(ethylene glycol)-diacrylate 
 ( peg - da , extralink , average mw = 3350 
 da ) were obtained from biotime inc .
this ha - sh / peg - da 
 hydrogel system previously has been characterized extensively .
dmso , l - cysteine , and papain papaya latex were obtained from sigma - aldrich 
 ( st . louis , mo ) .
primary antibodies used were human - specific anti - nuclei 
 antibody from millipore ( mab1281 , billerica , ma ) , anti - androgen receptor 
 from santa cruz biotechnology ( sc-816 , dallas , tx ) , anti - cleaved caspase-3 
 from cell signaling technology ( nb11089717 , danver , ma ) , and 
 anti - ki-67 from novus biologicals ( # 9664s , littleton , 
 co ) .
sylgard 184 poly(dimethylsiloxane ) ( pdms ) elastomer kit was from 
 dow corning ( midland , mi ) .
mda pca 183 
 and mda pca 118b cells 
 were routinely maintained as subcutaneous tumors in cb-17 scid mice 
 ( charles river ) .
all experiments for the propagation of pdx tumors 
 in mice were conducted under iacuc approval from the university of 
 texas md anderson cancer center .
the mda pca 183 xenograft was derived 
 from androgen - dependent prostate carcinoma , whereas the mda pca 118b 
 was derived from androgen receptor - negative castrate - resistant prostate 
 carcinoma . for this study ,
mda pca 183 
 and mda pca 118b pdxs of passage 12 and 8 respectively , were used . 
 on the day of harvest ,
tumor specimens were removed immediately , rinsed six 
 times with pbs , minced with a scalpel blade , and digested with accumax 
 enzymatic solution for 15 min at 37 c .
the enzyme solution was 
 inactivated with fbs , the resultant tumor slurry was filtered through 
 a 70 m cell strainer , and the filtrate was centrifuged .
the 
 supernatant was removed , the resulting cell pellet was resuspended , 
 and a cell count was performed .
custom - made pdms molds were made 
 by mixing sylgard 184 elastomer and cross - linker 10:1 ( v / v ) according 
 to manufacturer s instructions .
the liquid silicone solution 
 was centrifuged at 4 c/3000 rpm/5 min to remove bubbles and 
 poured into a square aluminum frame with a 1.5 mm - thick spacer .
an x-660 automated 
 co2 laser cutter ( universal laser systems , scottsdale , 
 az ) was used at 2.5% speed and 60% power to cut the slabs into 24 
  60 mm rectangles with multiple 6 mm diameter cylindrical cavities . 

the resultant molds were steam - autoclaved before each use and were 
 sealed onto sterile glass slides before being employed as molds for 
 hydrogel fabrication .
each cylindrical cavity could hold approximately 
 50 l of hydrogel . prior to pdx tumor culture
, ha - sh and 
 peg - da were solubilized at 10 and 20 mg / ml , respectively , in degassed 
 water per the manufacturer s instructions .
the solutions were 
 mixed at 4:1 ( v / v ) ratio , 35 l of the combined solution was 
 placed in each cavity of the silicone molds , and the mixture was allowed 
 to cross - link for 1 h at 37 c .
the bottom layer of acellular 
 hydrogel served as a  cushion layer  to prevent cell 
 clusters from settling out of the cell  hydrogel construct during 
 culture . following tumor harvest ,
were plated on 6-well plates at densities 
 determined to yield hydrogel constructs with 150,000 and 300,000 encapsulated 
 mda pca 183 and mda pca 118b cells respectively , per construct .
mda 
 pca 183 and mda pca 118b cells were cultured in dmem / f-12 supplemented 
 with 10% and 30% ( v / v ) fbs , respectively , in the presence of 100 u / ml 
 of penicillin and 100 g / ml of streptomycin . tumor cells then 
 were incubated at 37 c with 5% co2 for 2 days , after 
 which tumor cell aggregates that formed in suspension from all wells 
 were collected into sterile 15 ml tubes and gently centrifuged .
supernatant 
 was removed from each tube , and the remaining cell pellets were resuspended 
 in complete dmem / f-12 medium , combined , and split into microcentrifuge 
 tubes and centrifuged again .
notably , as these cells form clusters 
 in suspension , we could not perform cell counts immediately prior 
 to encapsulation . instead
, cell counting for the preparation of hydrogel 
 constructs was performed at the step of 2d culture in 6-well plates 
 ( immediately after the tumors were dissociated ) .
the resulting cell 
 pellets then were resuspended in solutions of ha - sh and peg - da ( 4:1 
 v / v ) as described above , and 25 l of the cell - hydrogel suspension 
 was immediately pipetted into each mold cavity , over the 35 l 
 cushion layer of cross - linked ha - sh / peg - da .
seeded hydrogels were 
 returned to the incubator for 45 min , then immersed in cell culture 
 medium and incubated overnight .
the following day , each mold cavity 
 was scored with a 26-gauge needle , cell - hydrogel constructs were transferred 
 into 24-well plates and submerged in culture medium .
the c4 - 2b bone metastatic 
 pca cell line was maintained in t - medium containing 5% fbs ( v / v ) and 
 2 mm l - glutamine in the presence of 100 u / ml of penicillin 
 and 100 g / ml of streptomycin .
for the docetaxel drug study , 
 50,000 cells were encapsulated within ha - sh / peg - da as described above 
 for the pdx tumors .
a lower cell density was used for the cell line 
 because high - density culture was associated with poor cell viability 
 ( data not shown ) .
cell - hydrogel constructs 
 ( n = 3 ) were maintained for 2 days before treatment 
 with docetaxel 
 for 3 days .
docetaxel was diluted in dimethyl sulfoxide ( dmso ) such 
 that the final concentration of dmso was 1% ( v / v ) in complete medium 
 across all drug concentrations .
morphology of the cells 
 encapsulated within 
 the hydrogel was monitored by differential interference contrast microscopy 
 at days 1 , 3 , 5 , and 7 postencapsulation using a nikon eclipse te300 
 inverted microscope and nis elements software ( nikon instruments , 
 melville , ny ) .
fluorescently labeled samples were imaged using a nikon 
 a1-rsi confocal microscope and images processed using the nikon nis - elements 
 ar software ( nikon instruments , melville , ny ) .
cell viability was assessed using the 
 live / dead viability / cytotoxicity kit as per the manufacturer s 
 instructions .
briefly , cell - hydrogel constructs at the designated 
 time - points were incubated in 2 m calcein - am and 4 m 
 ethidium homodimer-1 in pbs for 30 min at 37 c before confocal 
 imaging .
cell - hydrogel constructs ( n = 3 or 4 ) were collected into individual microcentrifuge 
 tubes at the designated time - points , flash - frozen using liquid nitrogen , 
 and stored at 80 c .
frozen samples then were lyophilized 
 overnight and digested in pbe buffer ( 0.10 m na2hpo4 and 0.010 m na2edta in demineralized water at 
 ph 6.5 ) containing 125 g / ml papain in the presence of 14.5 
 mm l - cysteine at 65 c overnight .
the digested samples then were sonicated using a probe 
 sonicator , and the liquid supernatant was assayed using the quant - it 
 picogreen dsdna quantification assay as per the manufacturer s 
 instructions .
excitation and emission wavelengths of 485 and 528 nm , respectively , 
 were used to measure the fluorescence ( flx800 fluorescence microplate 
 reader ; biotek instruments ) .
cell - hydrogel constructs were washed 
 with pbs and fixed with 4% ( v / v ) paraformaldehyde for 10 min at room 
 temperature .
after fixation , constructs were washed with pbs and stored 
 at 4 c until staining .
constructs were immersed in 0.2% ( v / v ) 
 triton x-100 for 5 min at room temperature to permeabilize cells , 
 then blocked with 500 l of 3% ( w / v ) bsa and 0.2% triton x-100 
 in pbs at 4 c overnight .
all antibodies were diluted at 1:200 
 in 3% bsa and 0.2% triton - x-100 in pbs .
antibody staining was performed 
 using 200 l of the mixed solution to each sample , which were 
 placed on a rocking platform shaker at 4 c overnight .
samples 
 were washed with pbs before adding fluorophore - labeled secondary antibodies 
 directed against the appropriate host .
secondary antibodies were diluted 
 1:500 in 3% bsa and 0.2% triton - x-100 in pbs , and 200 l of 
 that solution was added to each sample .
dapi ( 5 g / ml ) 
 was added to each sample at room temperature for 5 min .
when phalloidin 
 was used , it was diluted 1:20 in pbs , and 100 l of that mixture 
 was added to each sample for 15 min .
in initial experiments , following tumor digestion , 
 we encapsulated the entire pdx cell population directly into hydrogels . 

when we did so , a large number of dead cells was transferred to the 
 hydrogels as observed after 1 day in 3d culture ( data not shown ) . 

these dead cells likely were generated during the tumor harvest and 
 digestion , and also contain mouse - derived cells that die immediately 
 within the hydrogels . as the presence of high numbers of dead cells 
 would complicate any biochemical assays that were envisioned , we sought 
 to develop an alternative procedure designed to encapsulate cells 
 with high viability ( figure 1a ) .
( a ) mda pca 
 183 and mda pca 118b pdx tumors ( indicated by red arrows ) that were 
 grown subcutaneously in scid mice were harvested , then mechanically 
 and enzymatically digested .
the tumor slurry was subsequently plated 
 onto 6-well plates and cultured for 2 days during which the majority 
 of mouse - derived cells ( red ) attached .
the resulting tumor aggregates 
 that formed ( blue ) then were encapsulated within ha - sh / peg - da hydrogels 
 and cultured for up to 14 days .
( b ) dissociated tumor cells plated on tissue culture plastic 
 for 2 days formed multicellular clusters in suspension with other 
 cell types adhering to the plastic surface .
( c ) mixture of dapi - stained 
 ( blue ) cells within tumoroids and adherent cells after 2 days in culture . 

scale bars = 100 
 m . in optimizing the culture conditions 
 for the pdx pca cells
, we 
 observed that most of the mda pca 183 and mda pca 118b pca cells that 
 had undergone mechanical and enzymatic digestion and been plated onto 
 6-well plates formed multicellular aggregates in suspension after 
 2 days in culture , presumably reflecting their characteristic poor 
 adherence onto tissue culture plastic ( figure 1b ) .
additionally , during this period , we noted that a population 
 of cells in the tumor slurry ( shown in red in figure 1a ) adhered to the tissue culture plastic surface .
leveraging 
 this phenomenon , we collected the aggregates in suspension ( leaving 
 behind the adherent cells ) and found that the process of gentle centrifugation 
 resulted in the removal of dead cells . given that the mda pca 183 
 and mda pca 118b tumors were grown as subcutaneous tumors in mice , 
 they carry along with them a subpopulation of mouse - derived cells 
 in addition to the human pca cells .
indeed , we found that among the 
 poorly adherent pca cells that formed multicellular aggregates in 
 suspension after 2 days on tissue culture plastic , the cells that 
 had adhered stained positive for vimentin ( figure 1c ) . henceforth , we employed this pre - encapsulation 2d culture 
 method , which not only removes dead cells , but also enriches the pdx 
 tumor population via the depletion of mesenchymal cells .
a summary 
 of the process used to form 3d pdx tumoroids within the ha - sh / peg - da 
 hydrogel is illustrated in figure 1a . in the first week 
 after encapsulation ,
mda pca 183 cells were maintained as large tumoroids 
 with most having diameters between 60100 m .
mda pca 
 118b cells were maintained as smaller tumoroids , typically smaller 
 than 60 m in diameter ( figure 2a and supplementary figure 1 ) .
cells in tumoroids were 
 in close contact in multicellular clusters , observed via staining 
 with phalloidin for f - actin ( figure 2b ) . to 
 demonstrate that human pca cells comprise the 3d pdx tumoroids , we 
 stained the encapsulated cells for epithelial cell adhesion molecule 
 ( epcam ) , an established marker for epithelial cells .
as expected , 
 both the mda pca 183 and mda pca 118b 3d pdx tumoroids stained positive 
 for epcam ( figure 2c ) , indicating that the 
 tumoroids form from self - sorting epithelial cells despite the presence 
 of mesenchymal cells in the original xenograft tumors .
additionally , 
 we also confirmed this by probing for the human - specific anti - nuclei 
 antibody and found that the majority of cells within the tumoroids 
 stained positive ( data not shown ) , indicating that the 3d pdx tumoroids 
 form from largely human cells .
next , we asked if the pca cells in 
 tumoroids retained their androgen receptor activation status . probing 
 specifically for the androgen receptor , we found that while the androgen 
 receptor was mainly localized in the nucleus of cells in the mda pca 
 183 tumoroids , nuclear localization of the receptor was not observed 
 in the mda pca 118b cells as would be expected in vivo .
generation 
 of pdx tumoroids encapsulated within 3d ha - sh / peg - da 
 hydrogels .
( a ) mda pca 183 and mda pca 118b cells remained as multicellular 
 tumoroids post - encapsulation , over 1 week in culture .
cells were stained with dapi ( blue ) or phalloidin ( green ) ; 
 a merged image is shown on the right panel .
( c ) hydrogel - encapsulated mda pca 183 and mda pca 118b cells were 
 stained with dapi ( blue ) , and with antibodies against epcam ( green ) 
 and the androgen receptor ( red ) . while the mda pca 183 cells exhibited 
 nuclear localization of the androgen receptor ( indicated by yellow 
 arrows ) , the mda pca 118b cells stained negative for nuclear localization 
 of the androgen receptor .
encapsulation 
 of the nonadherent tumoroids into ha - sh / peg - da hydrogels maintained 
 cell  cell contacts for at least 2 weeks in culture .
given that 
 these pdx pca tumors exhibit poor viability in vitro on 2d , we next 
 investigated whether the encapsulated pdx tumoroids remained viable 
 within the hydrogels over time using the live / dead viability / cytotoxicity 
 assay .
as shown in figure 3 , cells in both 
 the mda pca 183 and mda pca 118b tumoroids were predominantly viable 
 at days 1 , 5 , and 14 . remarkably , pdx cells had the highest survival 
 when they had formed large clusters with other cells .
dead or dying 
 cells in each hydrogel were observed to be one of two types : either 
 single cells that had not aggregated with other cells to form multicellular 
 clusters or cells on the periphery of each cluster ( particularly prominent 
 in figure 3b , day 14 ) . beyond viability , to 
 establish if the 3d pdx tumoroids demonstrate tumor growth characteristics 
 necessary for the model to serve as a drug - testing platform , particularly 
 for drug candidates that target actively dividing cells , we monitored 
 their growth over 1 week in culture , using dna content as a surrogate 
 measure of cellularity .
notably , the overall cellularity of the mda 
 pca 118b constructs was lower than that of the mda pca 183 because 
 the mda pca 118b cells formed smaller clusters , therefore the retrieval 
 of cells for hydrogel encapsulation was less efficient than from the 
 mda pca 183 cultures .
this is apparent from the finding that even 
 though a higher theoretical seeding density ( 150,000 and 300,000 cells 
 per construct for mda pca 183 and 118b , respectively ) was employed 
 for the mda pca 118b constructs , the average initial dna content of 
 the mda pca 118b constructs still was lower than that of the mda pca 
 183 constructs ( day 1 , figure 4a ) . in analyzing 
 the cellularity of
the 3d pdx constructs over time , while dna content 
 remained constant with no significant difference over time for the 
 mda pca 183 tumoroids , there was a significant decrease in cellularity 
 for the mda pca 118b from day 1 to 5 , after which an increase in cellularity 
 was observed ( figure 4a ) . probing the 3d pdx 
 tumoroids for ki-67 and cleaved caspase-3 , markers 
 of proliferation and apoptosis , respectively , we found that differences 
 in the proportion of proliferative and apoptotic cells at day 5 in 
 culture was not apparent for either mda pca 183 or 118b ( figure 4b , c ) .
panels in ( a ) and ( b ) show mda pca 183 and 118b after 1 , 5 , and 14 
 days in culture , respectively . cells were stained with calcein - am 
 ( green , left panel ) or ethidium homodimer-1 ( red , middle panel ) .
( a ) 
 average cellularity of the 3d mda pca 183 and 118b constructs over 
 1 week in culture .
upper panel of ( b ) and ( c ) shows the mda pca 183 
 and mda 118b tumoroids , respectively , stained with dapi ( left panel , 
 blue ) and antibodies against ki-67 ( middle panel , 
 red ) .
lower panel of ( b ) and ( c ) shows the mda pca 183 and mda pca 118b 
 tumoroids , respectively , stained with dapi ( left panel , blue ) and 
 antibodies against cleaved caspase-3 ( middle panel , red ) the right 
 panel shows a merged image where green indicates f - actin .
having demonstrated 
 that the encapsulated 3d pdx tumoroids are ( 1 ) made up of viable pca 
 cells , ( 2 ) maintain in vivo - like androgen receptor distribution , and 
 ( 3 ) are proliferative in the hydrogels , we next determined the suitability 
 of this in vitro platform for drug testing of primary pca cells . given 
 the inherent differences in origin between the mda pca 118b and mda 
 pca 183 pdx models , we hypothesized that they would demonstrate a 
 differential sensitivity to chemotherapeutic drugs . to test this hypothesis
, 
 we exposed the 3d mda pca 183 and mda pca 118b tumoroids to docetaxel , 
 currently part of the first line regimen to treat patients with castrate - resistant 
 metastatic pca .
surprisingly , we found that not only was there no 
 overall difference in docetaxel sensitivity between the 3d mda pca 
 183 and mda pca 118b tumoroids , no significant reduction in cell number 
 was detected within the range of docetaxel concentrations tested for 
 either 3d tumoroid systems ( figure 5a ) .
to 
 confirm these findings , we performed the live / dead viability / cytotoxicity 
 assay and found that cells were largely viable even at the highest 
 docetaxel concentration ( figure 5b ) .
additionally , 
 there were no apparent differences in the proportion of apoptotic 
 cells as indicated by cleaved caspase-3 staining at the various docetaxel 
 concentrations tested ( supplementary figure 2 ) . to demonstrate that the lack of cell - kill was cell source - dependent , 
 we similarly exposed hydrogel - encapsulated cells from a bone metastatic 
 prostate cancer cell line , c4 - 2b , to docetaxel .
interestingly , a significant 
 decrease in cellularity was already apparent between 1 and 10 nm ( figure 5a , b ) .
this finding was corroborated by an observed 
 increase in the proportion of ethidium homodimer-1-stained cells with 
 increasing concentration of docetaxel ( figure 5b ) .
notably , given that viable c4 - 2b cells were present at 10,000 
 nm as observed by calcein - am staining , it is likely that the decrease 
 in the proportion of calcein - am - stained c4 - 2b cells due to drug cytotoxicity 
 could not be clearly discerned when compared to controls .
we also 
 showed that the pdx cells were readily killed with 1 m sodium azide ; 
 thus , it was likely not an issue of drug accessing the pca cells ( data 
 not shown ) .
( a ) average cellularity 
 of 3d mda pca 183 and 118b constructs after exposure to increasing 
 docetaxel concentrations , compared to the c4 - 2b cell line , also hydrogel - encapsulated . 

c4 - 2b cells exhibited significantly lower resistance to docetaxel 
 as compared to both pdxs ( p < 0.05 ) .
( b ) viability 
 of cells after exposure to 0 , 10 , or 10,000 nm docetaxel , assessed 
 by calcein - am staining ( green , left panels ) and ethidium homodimer-1 
 ( red , middle panels ) .
in 
 this study , we assessed a 3d ha - based hydrogel system for the 
 culture of primary pdx bone metastatic pca tumors , well - known to exhibit 
 poor viability when cultured on tissue culture plastic in vitro . given 
 the ubiquity of ha in the bone marrow extracellular matrix and our 
 previous studies that demonstrated the feasibility of using 3d ha - based 
 hydrogels to culture poorly adherent bone metastatic pca cell lines , 
 we hypothesized that these biologically active hydrogels would serve 
 as excellent matrices to support the long - term culture of primary 
 pdx pca tumor tissue in vitro .
the inability of bone metastatic 
 pca cells , whether derived from 
 cell lines or primary pdx tumor tissue , to grow in 2d culture is an 
 established phenomenon that indicates the lack of critical components 
 from the bone microenvironment upon which these highly adapted cells 
 depend . in optimizing the culture conditions for the 3d pdx pca cells
, 
 we found that the cancer cells failed to adhere to tissue culture 
 plastic , as was expected .
instead , these cells aggregated to form 
 multicellular aggregates in suspension , where the formation of cell 
cell 
 contact is a likely mechanism employed by the pca cells to adapt to 
 the loss of native cell - matrix interactions . indeed , as proposed by 
 shen and kramer , cancer cells are capable of undergoing  synoikis  , 
 a term that was used to describe the avoidance of apoptosis by relying 
 on intercellular adhesions in the absence of cell  matrix interactions .
focusing specifically on squamous cell carcinoma 
 cells , the authors demonstrated that e - cadherin - mediated cell  cell 
 contact resulted in the generation of compensatory survival signaling 
 via the epidermal growth factor receptor .
interestingly , a similar observation was made by a landmark report 
 by kondo et al .
cell 
 interactions were necessary for the survival of primary patient - derived 
 colorectal cancer cells in suspension culture . in optimizing the culture method for the primary 
 pdx pca cells , 
 we leveraged the ability of the cancer cells to spontaneously aggregate 
 to form multicellular clusters in suspension as a means to reduce 
 the presence of  contaminant  cells such as dead cells , 
 including those that were originally from the tumor tissue itself 
 ( in regions of necrosis ) and those that were generated during the 
 process of tumor digestion .
we found that the application of gentle 
 centrifugation to retrieve the pca aggregates in suspension after 
 2 days on tissue culture plastic was an effective method to separate 
 the pca cells from dead cells in suspension prior to encapsulation 
 within the hydrogel .
given that the pdx pca cells were maintained 
 in mice prior to harvest , the cancer cells were also likely  contaminated  

with mouse - derived cells ( such as blood cells , endothelial cells , 
 or fibroblasts ) , which consequently introduces a source of heterogeneity 
 and complicates downstream biochemical studies .
furthermore , considering 
 the importance of the tumor microenvironment in influencing tumor 
 behavior , the presence of mouse - derived stroma may confound studies 
 evaluating species - specific stroma - targeting drug candidates , hence 
 making it a major hindrance in current pdx tumor models .
indeed , while the pca cells formed aggregates 
 in suspension , a population of cells was consistently observed to 
 adhere to the tissue culture plastic surface .
for example , in mda 
 pca 183 cultures , we found that the adherent cells expressed vimentin 
 and originated from mouse tissue ( figure 1c ) . 
 while further characterization is necessary to determine the actual 
 yield of the cancer cells
, these results , in conjunction with the 
 observation that epcam , an epithelial cell marker , was near - ubiquitously 
 expressed in all of the hydrogel - encapsulated pdx clusters ( figure 2c ) , highly suggest that the pre - encapsulation 2d 
 culture serves as a desirable selective method to generate viable 
 and enriched pdx pca cells in 3d . in this study , we optimized 
 the culture of pdx pca cells in vitro , 
 which to our knowledge is unprecedented . while robust growth was limited 
 by a balance between proliferation and apoptosis , viability of the 
 hydrogel - encapsulated pca cells
was notably well maintained even up 
 to 14 days in culture ( figure 3 ) .
ten to 20% 
 of cells within the 3d pdx tumoroids stained positive for ki-67 at day 5 in culture ( figure 4b , c ) , indicating that a subset of cells was still progressing through 
 the cell cycle .
comparing the proportion of proliferative cells within 
 the 3d mda pca 118b pdx tumoroids to their in vivo counterparts , we 
 found that our estimate of ki-67-positive cells is 
 well within the range of that in vivo .
we are currently modifying the ha - based hydrogel to study the effects 
 of ecm moieties and other cell types on the growth behavior of the 
 pdx pca tumoroids in 3d culture . with the shift in focus from 
 traditional chemotherapy to biologically 
 targeted approaches based on a mechanistic understanding of pca biology , 
 preservation of salient features of
the original tumor is a critical 
 criterion in evaluating the success of this culture system as a drug - testing 
 platform . to this end
the androgen receptor is an important modulator 
 for prostate growth and currently the primary therapeutic target in 
 pca .
additionally , this nuclear receptor 
 regulates psa expression , which is used clinically as a biochemical 
 marker to track the progression of pca and response to therapy .
predominantly cytoplasmic in the absence of 
 ligands , ligand binding to the androgen receptor results in conformational 
 changes , nuclear translocation of the ligand - bound receptor , and transcriptional 
 activity . mirroring the in vivo tumor , 
 detection of nuclear androgen receptor in the 3d mda pca 183 tumoroids 

indicates that the ha - sh / peg - da hydrogel system is capable of maintaining 
 critical androgen receptor signaling in these androgen - dependent cells . 

similarly , nuclear androgen receptor was not observed in the 3d mda 
 pca 118b tumoroids as would be expected in in vivo , indicating that 
 these 3d pdx models may have utility as drug - testing platforms evaluating 
 androgen receptor - targeting agents currently under intense clinical 
 investigation .
drug discovery and screening 
 has historically relied on cancer 
 cell lines , which have generated a wealth of knowledge in cancer biology . 

however , the dichotomy in drug efficacy between preclinical and clinical 
 testing is increasingly apparent , primarily attributed to the genetic 
 and epigenetic changes that accrue when cells acclimatize to the in 
 vitro culture environment in extended periods of cell culture . indeed , gillet et al .
evaluated the multidrug 
 resistance transcriptome of six cancer types and found no correlation 
 between clinical samples and established cell lines , underscoring 
 the need for new in vitro cancer models and primary tumor models .
hypothesizing that inherent differences exist 
 between traditional pca cell lines and pdx pca tumors , we evaluated 
 the response of the 3d pdx pca tumoroids ( mda pca 183 and 118b ) to 
 docetaxel and compared their response to the c4 - 2b bone metastatic 
 prostate cancer cell line
. docetaxel is a microtubule stabilizer and 
 is currently part of the first - line standard treatment for metastatic 
 castrate - resistant pca .
we found that 
 despite the differences between the mda pca 183 and 118b cells in 
 patient origin , their overall drug sensitivity to docetaxel was similar . 

given that both pdx - derived cultures showed a low number of proliferating 
 cells and that docetaxel targets actively dividing cells , it thus 
 was unsurprising that both demonstrated similar resistance to the 
 drug in 3d culture .
however , interestingly , the 3d pdx pca cells exhibited 
 a significantly higher resistance to the drug as compared to the c4 - 2b 
 cells .
it is unlikely that the increased resistance is due to impaired 
 diffusion of the drug into the hydrogel since it demonstrated clear 
 efficacy against the hydrogel - encapsulated c4 - 2b cells
. while results 
 from this drug study are promising , studies are ongoing to establish 
 clinical relevance and understand the mechanisms underlying this difference . with the increasing awareness
that irreversible biological changes 
 occur when conventional cell lines are established in vitro , beyond 
 prostate cancer , there is general shift toward the use of pdx tumor 
 models for cancer research .
however , their 
 utility depends on the ability to manipulate the tumor cells ex vivo 
 prior to implantation .
given the alterations 
 in biological properties associated with 2d culture , a few groups 
 have taken steps to optimize methods to reliably grow primary tumor 
 cells in 3d .
one such example is in colorectal cancer , where it has 
 been shown that primary colorectal cancer cells can be cultured as 
 3d spheroids or colospheres in vitro , enabling the manipulation of 
 the cancer cells before engraftment in vivo for controlled studies . in our current study , we demonstrate for the first time that primary 
 bone metastatic pca cells can similarly be cultured in vitro with 
 the use of a 3d ha - based hydrogel system .
lastly , this 3d pdx model 
 may ultimately be adapted to a rapid and high throughput platform 
 for assessing drug efficacy , rational drug combinations , and development 
 of predictive biomarkers for novel targeted therapies , while reducing 
 the need for low throughput animal hosts . as an example
, we showed 
 here that 3d pdx pca cells exhibited an increased resistance to docetaxel 
 as compared to a standard cell line commonly used in pca research . 

ultimately , it is envisioned that the use of 3d pdx models may greatly 
 accelerate the advancement of novel drug candidates together with 
 predictive biomarkers that enable patient selection when translated 
 into early phase clinical trials .
on the basis of previous success culturing bone metastatic pca 
 cell lines using 3d ha - based hydrogels , we demonstrate for the first 
 time that  never in 2d  pdx pca tumors can be cultured 
 in vitro and maintained for at least 2 weeks .
the resulting hydrogel - encapsulated 
 3d pdx tumoroids retained viability , proliferative capacity , and the 
 androgen receptor expression , indicating that this novel 3d pdx model 
 may serve as a valuable platform for drug development .
while it has 
 yet to be tested , this system promises the possibility of culturing 
 tumor tissue directly from the patient for rapid drug screening , thereby 
 eliminating the  middle mouse  and its associated problems , 
 a major leap toward personalized medicine .
 the lack of effective 
 therapies for bone metastatic prostate cancer 
 ( pca ) underscores the need for accurate models of the disease to enable 
 the discovery of new therapeutic targets and to test drug sensitivities 
 of individual tumors . to this end , 
 the patient - derived xenograft ( pdx ) 
 pca model using immunocompromised mice was established to model the 
 disease with greater fidelity than is possible with currently employed 
 cell lines grown on tissue culture plastic . 
 however , poorly adherent 
 pdx tumor cells exhibit low viability in standard culture , making 
 it difficult to manipulate these cells for subsequent controlled mechanistic 
 studies . to overcome this challenge , we encapsulated pdx tumor cells 
 within a three - dimensional hyaluronan - based hydrogel and demonstrated 
 that the hydrogel maintains pdx cell viability with continued native 
 androgen receptor expression . 
 furthermore , a differential sensitivity 
 to docetaxel , a chemotherapeutic drug , was observed as compared to 
 a traditional pca cell line . 
 these findings underscore the potential 
 impact of this novel 3d pdx pca model as a diagnostic platform for 
 rapid drug evaluation and ultimately push personalized medicine toward 
 clinical reality . 
Introduction
Materials
and Methods
Results
Discussion
Conclusions