prostate cancer is the most   frequent tumor in men , afflicting african american males to a greater degree than caucasians .
morbidity and mortality are mainly attributable to metastasis ; yet the mechanisms associated with progression are largely unknown .
localized carcinomas are readily removed surgically , but once a tumor has established metastases , current therapies are not curative and prolong survival by only a few years .  
metastasis occurs through a multistep process , where metastatic cells must intravasate local tissues and enter into and survive in the blood stream .  
these cells then extravasate into the secondary tissue and initiate and maintain micrometastases at distant sites , with the end result being the development of a metastatic tumor     [ 1 , 2 ] . during each step of this process
, cancer cells exhibit transdifferentiation properties that allow both the spatial and temporal expression of epithelial and mesenchymal properties in response to microenvironment signals and its own basic survival needs ( e.g. , motility and invasion versus proliferation ) .   
thus , a model of cellular transitions , as opposed to a continual progression to permanent differentiation state , is emerging as a significant mechanism during metastasis
. a greater understanding of these mechanisms will result in clinical improvements and a better control of the metastasis process .
epithelial - mesenhymal transition ( emt ) was first described during development     [ 3 , 4 ] ; however an emt - like phenotypic change has been observed in a number of solid tumors     [ 57 ] .  
this transition is typically characterized by a loss in e - cadherin and cytokeratin expression .
emt in cancer , as in development , is associated with an increase in cell proliferation     [ 8 , 9 ]   and the acquisition of a mesenchymal phenotype that includes vimentin , n - cadherin , and osteopontin expression . in both   normal development emt and cancer - associated emt , the loss of e - cadherin is critical to the differentiation and maintenance of the epithelial phenotype   and provides a structural link between adjacent cellular cytoskeletons , which is important for tissue architecture .
cells that have undergone emt ( e - cadherin   negative mesenchymal cells ) subsequently become more migratory and invasive   and proceed to traverse underlying basement membranes , with an acquired ability to intravaste the surrounding local tissue and gain access to vascular conduits . as such , the loss of e - cadherin is rate limiting for emt     [ 10 , 11 ] .
recent reports from this laboratory and others   have described a mesenchymal to epithelial reverting transition ( mert ) to occur , where mesenchymal - like prostate cancer cell lines reexpress e - cadherin to become epithelial - like , and reestablish cellular adhesion during colonization within the liver tumor microenvironment     [ 12 , 13 ] .  
these findings are shared in clinical metastases of various cancer origins including breast , colon , and bladder , where robust membrane expression of e - cadherin was observed , and the paired more differentiated primary tumors were e - cadherin negative     [ 6 , 14 ] .  
thus , a reversion of the mesenchymal phenotype appears to be important in latter stages of metastasis .
numerous studies have shown that the underlying influence of these cellular transitions is a consequence of tumor - stromal interactions     [ 15 , 16 ] .
coculture studies have found that the survival and proliferation of cancer cells are intimately linked to the soluble factors in the microenvironment , such as egf , tgf- ,   igf - l that contribute to survival and the subsequent formation of macrometastasis     [ 1720 ] .  
however , these factors are not likely to have a direct effect during initial metastatic colonization , and thus heterotypic and homotypic cellular adhesion has been proposed to provide the necessary survival signals for successful colonization     [ 21 , 22 ] .
current state - of - the - art technology does not provide the necessary resolution to determine at the single cell level in patients or experimental in vivo systems , individual cells that have successfully colonized the secondary site .
however , numerous reports have firmly established that cancer - stromal interactions in vitro or in three - dimensional ( 3d ) assays accurately mimic the drug sensitivity / resistance behavior of those cells found within solid tumors in vivo in a preclinical or clinical setting     .  
thus , we employed a novel coculture assay to determine the cellular plasticity of cancer cells promoted by the bone stroma   and the effect of tumor - stromal interactions on irradiation therapy in prostate cancer .
the arcap model is the only robust prostate cancer bone metastatic model which demonstrates epithelial to mesenchymal   transition(emt ) .  
the arcap progression model consists of arcape ( epithelial ) and arcapm ( mesenchymal ) , where the arcape cells have a bone metastatic potential of 12.5% and the arcapm cells have a bone metastatic potential of 100% .
the arcape and arcapm cells express the classical markers of emt     [ 24 , 25 ] .   
herein we present findings that arcapm cells undergo mert when cocultured within the bone microenvironment in 3d and 2d cultures .  
additionally , arcape cells that retained an epithelial phenotype exhibited a measurable growth advantage and retained ability to form colonies , however only under coculture conditions with bone stroma .  
furthermore , blocking the ability of arcape or arcapm cells    from e - cadherin - mediated cell - cell adhesion   or integrin alpha v beta - associated adhesion significantly affected arcap cell survival within bone stroma and sensitized these cells to radiation treatment .
the human prostate cancer cell lines , arcape , arcapm , the hs-27a bone stromal cells ( atcc , manasss , va ) and the pt - n or pt - c human prostate stromal cell .  
isolation and characterization of the human prostate cancer rfp - arcap cell lines has been reported     .  
red fluorescent protein- ( rfp- ) transfected cells were maintained in g418 ( 350  mg / ml ) prior to experimentation .  
all cell lines were grown in a 5% co2 incubator at 37c in media consisting of t - medium ( invitrogen , carlsbad , ca ) supplemented with 5% ( v / v ) fetal bovine serum and 1% penicillin - streptomycin .
initial cocultures were performed as previously described     [ 12 , 13 ] with modifications .
cocultures consisted of 50  000 cells / cm     of   hs-27a bone marrow stromal cells and 2000 cells / cm prostate cancer cells .
cocultures were maintained in serum - free t - media   and plated on tissue culture dishes .
cells were plated at low densities in six - well plates for 24 hours and then were irradiated with the appropriate radiation dose .
twenty - four hours later , the media were changed and cells were incubated until they formed colonies having at least 50 or more cells .
seventeen days later colonies were rinsed with pbs , stained with methanol / crystal violet dye , and counted .
the colony formation ability was calculated as a ratio of the number of colonies formed , divided by the total number of cells plated , times the plating efficiency [ ( # of colonies formed  total # cells plated )  plating efficiency ] . for experiments with cocultures
, cells were initially incubated on a mat of stromal cells for 24 hours and radiated ; 4 hours later clonogenic assay was performed . for antibody - based experiments using anti - e - cadherin (
15  g / ml , decma or shep8 - 7 , sigma ) and anti - integrin alpha - v ( 20  g / ml , cnt095 ) antibody , cancer cells were treated with respective antibodies for 24 hours prior to plating them on a mat of stromal cells .
external beam radiation was delivered on a 600 varian linear accelerator ( varian medical systems , inc.palo alto , ca ) with a 6  mv photon beam .
a 40  40  cm field size was utilized and petri dishes were placed on 1.5  cm of superflab bolus .
monitor units ( mus ) were calculated to deliver the dose to a depth of dmax at a dose rate of 600  mu / min .
representative findings are shown for all experiments , which were performed in triplicate , repeated a minimum of three times .
recently , the arcap model has been described to closely mimic the patho - physiology of advanced clinical human prostate cancer bone metastasis     .  
these cells exhibit a cuboidal - shaped epithelial morphology with high expression of epithelial markers , such as cytokeratin 18 and e - cadherin .
the lineage - derived arcapm    cells have a spindle - shaped mesenchymal morphology and phenotype .
arcapm cells have decreased expression of e - cadherin and cytokeratins 18 and 19   but increased expression of n - cadherin and vimentin .
these cells have decreased cell adhesion and increased metastatic propensity to bone and adrenal glands     .
the morphologic and phenotypic changes observed in the arcapm cells closely resemble those of cells undergoing emt .   
previously , we have demonstrated a mesenchymal to epithelial reverse transition ( mert ) of metastatic prostate cancer cell lines within an experimental coculture model and confirmed in patients with liver metastasis     [ 13 , 28 ] .  
our findings have recently been confirmed in prostate cancer bone metastasis where e - cadherin and -catenin were robustly expressed in late stage carcinomas     .   
therefore we sought to identify the significance of the bone microenvironment within the experimental arcap model .   to assess cellular plasticity of the arcap emt model , we coultured arcap cells with hs-27a cells in 3d rwv ( rotary wall vessel ) system for 3 days .  
arcape   cells formed larger prostate organoids than arcapm cells   ( data not shown ) .   upon immunohistochemical examination of organoids , we observed that both arcape and arcapm express e - cadherin and lack n - cadherin expression ( figure 1(a ) ) .   to further examine the influence of tumor - stroma interactions over a multiday period we utilized a similar 2d cocultures method .
utilizing immunoctyochemical analysis , we observed a lack e - cadherin and robust n - cadherin staining after 1 day in both arcape and arcapm cocultures .
however by day 4 , both arcape and arcapm   cells formed tumor nest that express e - cadherin and lack n - cadherin staining ( figure 1(b ) ) .   
it is worthy to note that arcapm    tumor nest appeared to develop at much smaller extent , compared to arcape cocultures .   
since arcape cells formed larger tumor nest and spheroids when cocultured with hs-27a cells compared to arcapm cells , we sought to further assess if hs-27a cells preferentially stimulated the growth of arcape cells versus arcapm cells .  
utilizing gfp - transfected hs-27a bone marrow stromal cells and rfp - transfected arcape   or arcapm   cells ( figure 2(a ) ) , we examined the proliferative ability of arcap cells in homotypic and coculture conditions .
growth of rfp - transfected arcape and arcapm   cells , respectively , was quantified by relative fluorescent units ( rfu ) of transfected cell lines over a 6-day period in homotypic cultures and coculture conditions ( figure 2(a ) ) .   
as previously reported , homotypic cultured arcapm shows significant growth compared to arcape    homotypic cultures ; however cocultures reversed this trend with arcape   cells demonstrating the most significant growth ( figure 2(b ) ) .
although arcapm cells have a higher plating efficiency than arcape cells , arcape cells exhibited an 8-fold increase in their ability to form colonies after coculture compared to 1.35-fold increase of cocultured arcapm   cells ( figure 2(c ) ) .
phase - contrast microscopy of colonies after coculture shows that arcapm colonies appear loosely adherent , while arcape cells are compact and interact physically with few of the bone stromal fibroblast ( figure 2(d ) ) .   taken together , these results demonstrate that arcapm    cells reexpress e - cadherin when grown with bone stromal cells for longer periods . additionally , arcape cells which have high levels of e - cadherin gain enhanced growth and self - renewal ability when cocultured with bone stromal cells .
mesenchymal cancer cells have been thought to be more tumorigenic , aggressive , and resistant to treatments when compared to epithelial cancer cells .  
a similar trend was observed in both arcape and arcapm cells after ( 4  gy ) irradiation treatment .
arcapm   homotypic cancer cells are more resistant to radiation treatment compared to arcape homotypic cancer cells ( figure 3(a ) )
.   however , arcapm    cocultures did not affect the radiation sensitivity of arcapm    cancer cells .  
the highly sensitive arcape   cells exhibit a significant increased resistance to radiation therapy , up to 3-fold , as result of their interaction with bone stromal cells ( figure 3(a ) ,   p < .01 ) .    to further assess the role of the prostate stromal cells on tumor - stromal   interactions influencing arcap cellular behavior
, we cocultured paired prostate stromal fibroblasts isolated either from normal ( pt - n ) or from cancer - associated regions ( pt - c )     .  
again , arcape cells cocultured with ( pt - n ) or ( pt - c ) exhibited a 7-fold and 8-fold increase in colony formation , respectively ( figure 3(b ) , p < .01 ) .  
we also saw a similar trend in a growth analysis assay ( data not shown ) .   
however when measuring clonogenic ability after radiation treatment , arcape cells cocultured with either pt - n or pt - c had increased radiation resistance , with a 2-fold difference observed between homotypic cultured cells .  
although a significant increase in clonogenic formation was observed in pt - c versus pt - n cocultures ( p < .05 ) , this did not significantly effect    the radiation sensitivity of arcapm cells ( figure 3(c ) ) . taken together
, both bone and prostate stromal cell have a grown inductive effect on arcape   cancer cells and mediate radiation resistance ( up to 2 - 3 fold ) in epithelial cancer phenotype , but not in arcapm     mesenchymal cancer cells .
the importance of cell adhesion ( i.e. , cell - cell and cell - ecm adhesion ) on the survival of disseminated cancer cells has been well documented as a requirement for colonization and survival within the metastatic microenvironment     [ 3234 ] .  
therefore we utilized a well - known e - cadherin blocking antibody ( shep8 - 7 ) and a pan - integrin antibody ( cnt095 ) that targets human alpha - v - integrin   and also was shown to block prostate tumor growth within bone     .  
since arcape cells express high levels of the epithelial marker e - cadherin , and arcapm cells can be microenvironmentally induced to express e - cadherin , we tested whether either of these blocking antibodies would affect the colony forming ability of either arcape or   arcapm bone stroma - cocultured cells
.   pretreatment with e - cadherin antibody did not affect the colony forming capacity of either arcape or arcapm homotypic cultured cells ; however it   significantly reduced the   ability of arcapm-  ( p < .001 ) and arcape-  ( p <
additionally , e - cadherin blocking   antibody pretreatments   further increased sensitivity to radiation treatment of arcapm cells in homotypic and cocultured conditions , similarly   ( p < .01 ) .  
e - cadherin blocking antibody - pretreated arcape cells showed the most significant increased sensitivity to radiation treatment in homotypic compared cocultured conditions ( p < .001 ) , however a significant reduction in colony formation , to a lesser extent , was observed in arcape cocultured cells ( figure 4 , p < .01 ) .  
therefore , targeting e - cadherin limited both epithelial and mesenchymal cells ability to form colonies after coculture with bone stromal cells .
to determine the influence of intergin alpha v cell adhesion with bone microenvironment , we performed similar clonogenic formation assay .  
pretreatment with cnt095 antibody significantly decreased the clonogenic ability of both arcapm and arcape cells in homotyic cultures ( figure 5 , p < .001 ) .   additionally , cnt095 significantly decreased bone stroma - induced radiation resistance in cancer cells in both arcapm  ( p < .001 ) and arcape  ( p < .001 ) cancer cells , with the most significant reduction in   cocultured conditions ( p < .001 ) ( figure 5 ) . taken together , these results suggest that bone stroma - induced radiation resistance is mediated through both e - cadherin and integrin alpha v beta signaling   in epithelial and mesenchymal cells .   thus , e - cadherin and integrin alpha v beta appear to present novel targets for metastatic and radiation resistant cells .
it is well documented in prostate and others cancers that emt is associated with initial transformation from encapsulated to invasive carcinomas .  
the mesenchymal phenotype , which is required for dissemination , has been suggested to revert to an epithelial phenotype in distant metastasis     [ 13 , 14 , 29 , 36 ] .  
this has been evidenced in the primary tumors which lack e - cadherin expression and , showing nuclear -catenin expression , show strong membrane staining for both e - cadherin and -catenin in metastatic   liver   or bone microenvironment [ 28 , 29 ] .
we have previously shown , in commonly utilized prostate cancer cells lines du-145 and pc-3 ,   that   reexpression of e - cadherin and   reversion of the mesenchymal phenotype   is a rate limiting for metastatic seeding of primary rat hepatocytes .  
since bone metastasis is most prevalent in prostate cancers ,   we sought to extent these finding utilizing the arcap model , which is the first prostate cancer emt   model demonstrating histomorphological features and classical markers in a lineage - derived series of cells , to determine the functional relationship of this cellular transition .   whether this is accomplished through exposure to soluble growth factors or the bone microenvironment , the end result   decreased differentiation with increased metastatic potential     [ 25 , 27 , 37 ] .   
our initial results show that arcapm cells maintained in 3d rotary wall vessel ( rwv )   or 2d cocultures underwent mert when cocultured with hs-27a bone stromal cells , as shown through expression of e - cadherin and of   n - cadherin expression ( figures 1(a ) and 1(b ) ) .  
moreover arcape cells show a significant enhancement in colony formation ( 8 )   and significant growth pattern comparable to arcapm    ( 1.35 ) cocultures ( figures 2(b ) and 2(c ) ) .  
a recent report has shown through rfp cell tracking   that selected arcape clones after in vivo inoculation into the bone microenvironment    gives rise to both arcape and arcapm populations .
these findings coupled with our observed reversion of arcapm cells to arcape like cells   suggest that tumor - stromal - induced cellular plasticity   gives rise to   distinct populations of cancer cells within bone microenvironment , the mesenchymal phenotype and its kinetic characteristics ( motility / invasive ) , and the epithelial characteristics necessary for secondary tumor development .
the fact that the arcapm cells   have an increase propensity for metastasis compared to arcape cells   suggest that dissemination from the primary tumor mass requires the mesenchymal phenotype .
however a mesenchymal to epithelial transition is associated with initial metastatic seeding and subsequent formation of a cohesive tumor mass within the bone microenvironment .  
this hypothesis is supported in a bladder cancer model , where lineage - derived series of emt - transformed mesenchymal - like cells exhibit increased lung metastasis in vivo ;   however secondary tumor formation is predominantly enhanced by the presence of epithelial cells compared to mesenchymal cells     .   since epithelial reversion enhances the growth of tumor cells in bone microenvironment , and this
is observed in multiple experimental models and clinical metastases , there is a question of whether this transition is required for metastatic seeding and therefore an avenue for therapeutic intervention(s ) .   to gain insight into the importance of this reversion , we utilized ionizing radiation on arcape and arcapm homotypic and cocultured cells .  
our results show that arcape homotypic cultures when compared to arcapm homotypic cultures are more sensitive to radiation treatment ( figure 3(a ) ) .
however in the presence of bone or prostate stromal cells , arcape    cells gained increased radiation resistance , with increased proliferative and colony forming capacity ( figures 2(b ) and 2(c ) ) .
this phenomenon was not observed in the arcapm   cocultures . to determine the underlining causes of this observation
, we hypothesized that cell - cell interactions through e - cadherin or cell - ecm interactions through integrins   may mediate the stromal induced proliferative effect and radiation resistance in arcape    cancer cells . using e - cadherin neutralizing antibody ( shep8 - 7 ) and pan - anti - integrin alpha v antibody ( cnt095 )
, we were able to significantly block the stromal induced colony forming ability on arcape    cancer cells ( figures 4 and 5 ) . additionally , both antibodies significantly blocked the radiation resistance of arcape    in cocultured conditions   ( figures 4 and 5 ) .
the e - cadherin neutralizing antibody also had an effect on homotypic arcapm - radiated cells and arcapm    cells within cocultures ( figure 4 ) .
thus it appears that blocking bone stroma - induced reexpression of e - cadherin in arcapm in the presence of bone stromal cells reduced   the colony forming capacity of these cells   ( figure 4 ) .
the decreased radiation sensitivity of e - cadherin expressing cells compared to cells lacking e - cadherin expression has recently been demonstrated in a cocultured model of mcf-7 ( e - cad positive ) and mda - mb-231 ( e - cad negative ) cells with normal and radiation - induced senescent fibroblast , where radiation in mcf-7 cells showed enhanced resistance to radiation treatment compared to mda - mb-231 cells .
these findings are consent with our model of a reepithelization requirement within tumor microenvironment .   
cnt095 antibody was toxic to both arcapm    and arcape   homotypic and cocultured cells .  
additionally cnt095 increased radiation sensitivity , even to a greater extent than e - cadherin neutralizing antibody treatment ( figure 5 ) .
these findings are consistent with our results of ctn095 treatment that causes a significantly reduced number of   tumors generated by c4 - 2b cells , along with a concomitant increase of cortical bone in mice ( unpublished data ) .  
although c4 - 2b cells have not been observed to undergo emt , this would suggest that targeting the cell - ecm in vitro and in vivo could be limiting the cell cohesiveness necessary for metastatic tumor formation .   
e - cadherin neutralizing antibody ( shep8 - 7 ) has been shown to sensitize multicellular spheroids to microtubule binding therapies in the taxane family in ht29 human colorectal adenocarcinoma cells     .  
a more recent observation is that survival of androgen receptor - expressing differentiated prostate cells is dependent on e - cadherin and pi3k , but not on androgen , ar , or mapk .  
given the predominate role for pi3k in cell survival and reports that pi3k is rapidly recruited to cell membrane to stabilize e - cadherin junctions    and that pi3k activation requires integrin alpha v activity       suggests that pi3k is possibly responsible for the increased growth and colony formation gained within the tumor microenvironment .
thus in the absence of stimulating growth factors , it is possible that e - cadherin / pi3k or integrin alpha v / pi3k is involved in a signaling cascade that is initiated by the tumor microenvironment , at least during initial metastatic seeding .    in conclusion ,
our data demonstrate that the e - cadherin and integrin alpha nctional adhesive interaction is a possible adjuvant therapy avenue for patients treated with radiation .  
although   an in - depth in vivo exploration of targeting epithelial - like versus mesenchymal - like cells is necessary to translate these findings to the clinical situation , our results indeed raise critical questions as to how we view prostate cancer metastasis and subsequently target metastatic tumor cells for therapy .
additionally , we have generated an in vitro model , that closely mimics the clinical situation , to delineate in a stepwise manner the dynamic tumor - host interaction(s ) that promote cellular plasticity in the later stages of metastasis .
the identification of further key molecules driving mert in this system holds promise for the development of preventative and therapeutic strategies to minimize metastatic disease .
 hs-27a human bone stromal cells , in 2d or 3d coultures , induced cellular plasticity in human prostate cancer arcape and arcapm cells in an emt model .   cocultured arcape or arcapm cells with hs-27a , developed increased colony forming capacity and growth advantage , with arcape exhibiting the most significant increases in presence of bone or prostate stroma cells . 
 prostate ( pt - n or pt - c ) or bone ( hs-27a ) stromal cells induced significant resistance to radiation treatment in arcape cells compared to arcapm cells 
 . however pretreatment with anti - e - cadherin antibody ( shep8 - 7 ) or anti - alpha v integrin blocking antibody ( cnt095 )   significantly decreased stromal cell - induced radiation resistance in both arcape- and arcapm - cocultured cells . taken together 
 the data suggest   that   mesenchymal - like cancer cells reverting to epithelial - like cells in the bone microenvironment through interaction with bone marrow stromal cells and reexpress e - cadherin . 
 these cell adhesion molecules such as e - cadherin and integrin alpha v in cancer cells induce cell survival signals and mediate resistance to cancer treatments such as radiation . 
1. Introduction
2. Methods
3. Results
4. Discussion