
 cxcl16 , a newly identified chemokine , has been described in both transmembrane and soluble forms .
transmembrane cxcl16 ( tm - cxcl16 ) is expressed on the surface of macrophages , dendritic cells , and monocytes where it functions as an adhesion molecule for cxcr6-positive immune cells [ 1 , 3 ] .
tm - cxcl16 undergoes cleavage by the disintegrin - like metalloproteinases adam10 and adam17 before it is released in soluble form to the outside of the cell [ 4 , 5 ] .
soluble cxcl16 ( scxcl16 ) prompts migration of leukocytes expressing cxcr6 in a dose - dependent manner . because cancer cell migration and metastasis share patterns with leukocyte trafficking , attention has been focused on the role of cxcl16 in cancer progression .
cxcl16 is expressed in various cancers , including pancreatic , prostate , breast , colorectal , and nonsmall cell lung cancer [ 8 , 9 ] .
more importantly , lu et al . revealed a positive correlation between cxcl16 mrna expression and prostate cancer aggressiveness such that metastatic lesions expressed higher levels of cxcl16 mrna than the primary prostate tissues .
have shown that scxcl16 induces migration and proliferation of cxcr6-expressing prostate cancer cells [ 9 , 10 ] . moreover , matsushita et al . reported that high preoperative serous levels of scxcl16 were associated with liver recurrence and poor prognosis in patients with colorectal cancer .
immunohistochemical staining data from patients with colorectal or renal cancer correlated better long - term prognosis with stronger cxcl16 staining in cancer tissues [ 12 , 13 ] .
these limited reports imply different functions for cxcl16 depending on the location of its expression in cancer patients .
breast cancer is the most common malignancy and the second leading cause of cancer - related death in american women . despite survival rates having improved steadily since 1990
therefore , it is important to get a better understanding of the molecular mechanisms underlying breast cancer metastasis and to develop prognostic and therapeutic strategies . in this study , we explore the expression and function of cxcl16 in breast cancer cell lines that differ in aggressiveness .
the breast cancer cell lines sk - br-3 , mcf-7 , and mda - mb-231 were obtained from american type culture collection ( atcc ) ( rockville , md ) .
the noncancerous human mammary epithelial cell line mcf-10a was purchased from bioleaf biotech ( shanghai , china ) .
all cell lines were cultured at 37c in dulbecco 's modified eagle medium ( hyclone , waltham , ma ) supplemented with 10% fetal bovine serum , 100  units / ml penicillin , and 100  ug / ml streptomycin in a humid incubator with 5% co2 .
total rna was extracted by biozol reagent ( bioflux , tokyo , japan ) according to the manufacturer 's instructions .
less than 2  ug rna was reverse - transcripted into cdna using reverse transcriptase ( promega , beijing , china ) and oligo(dt)18 ( takara , dalian , china ) .
primers for cxcl16 were as follow : sense 5-ggcccaccagaagcatttac-3 and antisense 5-ctgaagatgccccctctgag-3. primers for glyceraldehyde 3-phosphate dehydrogenase were as follows : sense 5-gaaggtgaaggtcggagtc-3and antisense 5-gaagatggtgatgggatttc-3. pcr was performed with an iq4 multicolor real - time pcr detection system ( bio rad , hercules , ca ) using sso fast evagreen supermix ( bio rad ) .
pcr protocol was performed as follows : denaturing for three seconds at 98c followed by forty amplification cycles of annealing and extension at 55c for fifteen seconds .
normalized lysates ( 30  ug ) were separated by electrophoresis in 12% sds - page and electrotransferred onto polyvinylidene fluoride membrane ( pvdf membrane , millipore , billerica , ma ) .
the membrane was blocked with 5% nonfat milk in tris - buffered saline - tween ( tbst , ph 7.6 ) at room temperate for 1  h and incubated overnight at 4c with cxcl16 antibody ( abcam , cambridge , uk ) .
after three washes with tbst , the membrane was incubated with horseradish peroxidase- ( hrp- ) conjugated igg .
cells were trypsinized and 10 cells were incubated with pe - conjugated cxcl16 antibody ( r&d systems , minneapolis , mn ) in a dark room for 45  min .
after two washings with phosphate buffered solution ( pbs ) , expression of transmembrane cxcl16 in cells was analyzed with a becton dickinson facscan using a software facs express 3 ( de novo software , los angeles , ca ) .
cells were seeded with a volume of 200  ul ( 2,000 cells / well ) into 96-well plates ( corning ) .
every 24  h , mtt was added to the well with a final concentration of 0.5  mg / ml and subsequently incubated for 4  h at 37c .
supernate was discarded and 150  ul / well dmso was added . the optical densities ( od ) were measured at 490  nm with a microplate reader ( bio rad ) .
migration and invasion assays were performed using a transwell chamber ( 8 um pore size , millipore ) according to the manufacturer 's instructions .
cell culture inserts for the invasion assay were precoated with matrigel ( bd biosciences , bedford , ma ) for 4  h at 37c .
cells were seeded into the upper chamber , while 1  ml complete medium was added into the lower chamber as a chemotaxin .
after culture for 24  h , noninvading cells were removed with a cotton bud .
cells that migrated to the lower surface were fixed in 4% paraformaldehyde for 20  min and underwent giemsa staining .
five random fields were selected for cell counting under a light microscope ( 100 ; nikon , tokyo , japan ) .
caspase-3 was measured using the caspase-3 activity kit ( beyotime , nanjing , china ) .
appropriate cells were incubated with 30  ul lysis buffer on ice for 30  min and then subjected to centrifugation at 13,000  rpm for 5  min .
10  ul of supernatant was incubated with ac - devd - pna at 37c for 5  h. the od values were detected at 405  nm .
cells were collected with edta trypsin and washed twice with phosphate buffer solution ( pbs ) . in a 500  ul binding buffer ,
cells were stained with 5  ul annexin v and 5  ul 7 aad successively in the dark room .
apoptosis index is calculated by the percentage of cells with positive for annexin v and negative for 7-aad .
the day before transfection , mda - mb-231 cells were seeded into a six - well plate at a density of 10 cells / well .
after transfection in serum - free medium for 12  h , cells were further cultured in complete medium .
both cxcl16 and control plasmids were obtained from genepharma company ( shanghai , china ) .
five - week - old female nod / scid mice were purchased from beijing hfk bioscience co. , ltd .
cells were trypsinized into single cell suspension , and 3  10 cells were injected subcutaneously into the right - lower flank of each mouse .
tumor volumes were approximated using the formula v = [ ( /6 )  l  w ] , where l was the longest axis and w was the shortest axis .
all mice were sacrificed by cervical dislocation , and the tumors were removed and weighed .
all data are expressed as the mean  standard deviation ( sd ) and compiled using spss 16.0 ( spss , chicago , il ) .
one - way analysis of variance was used to determine statistical and significant differences between control and treated groups .
expression of cxcl16 was evaluated in mammary epithelial cell mcf-10a and in three breast cancer cell lines : sk - br-3 , mcf-7 , and mda - mb-231 . as shown in figure 1(a ) , cxcl16 mrna was highest in mcf-7 ( 3.038  0.436-fold ) , followed by sk - br-3 ( 1  0.548-fold ) , while the lowest expression was observed in mda - mb-231 ( 0.07    0.049-fold ) .
flow cytometry demonstrated that transmembrane cxcl16 was highest in mcf-7 and lowest in mda - mb-231 ( figure 1(b ) ) .
mcf-7 and sk - br-3 ( with 32.25  2.63 and 59.75    6.94 migrated cells ) showed less migration than mda - mb-231 cells ( 146.5    5.74 , p < 0.01 ) ( figure 1(c ) ) .
proliferation was the strongest in mda - mb-231 cells , while sk - br-3 cells and mcf-7 cells possessed less , though equal , proliferative capability ( figure 1(e ) ) . to further confirm the effect of cxcl16 on invasion , migration , and proliferation in these breast cancer cell lines
elevated cxcl16 levels were confirmed by western blot or flow cytometry ( figure 2(a ) ) .
consistently , cxcl16-overexpressing cells showed significantly decreased cell migration ( from 118  11.53 to 61.66  7.37 , figure 2(b ) ) and invasion ( from 84.0  5 to 48.66  4.04 , figure 2(c ) ) compared with the control mock - transfected cells .
however , mtt assay for cell proliferation revealed no difference between cells transfected with cxcl16 plasmids compared with the control mock - transfected cells ( figure 2(d ) ) .
in addition , apoptotic index was increased from 3.5  2.03% to 5.9  2.87% , and the activity of caspase-3 , a crucial enzyme in the apoptotic cascade , whose od value was enhanced from 0.65  0.032 to 0.89  0.121 , indicated that increased cxcl16 expression facilitated caspase-3-dependent apoptosis ( figure 2(e ) ) . meanwhile , to determine whether cxcl16 gene silencing would affect breast cancer progression , mcf-7 cells highly expressive for cxcl16 underwent stable knockdown of cxcl16 using shcxcl16 , scrambled shrna ( shnc ) , or liposomes only ( mock ) .
the efficiency of cxcl16 knockdown was confirmed by qrt - pcr , western blot , or flow cytometry which showed decreased cxcl16 expression in shcxcl16-transfected mcf-7 cells ( figure 3(a ) ) .
stable downregulation of cxcl16 expression in mcf-7 cells resulted in a significant increase in migration and invasion ( figures 3(b ) and 3(c ) ) , but not proliferation ( figure 3(d ) ) .
as shown in figure 4(a ) , cxcl16-overexpressing mda - mb-231 showed delayed tumor progression as compared to mda - mb-231 or the positive control ( mcf-7 ) ( p < 0.05 ) .
tumor volumes and weights for cxcl16-overexpressing groups were accordingly found to be significantly reduced ( p < 0.05 ) ( figure 4(b ) ) .
cxcl16 is a novel chemokine first cloned by maltoulin in 2000 . like other chemokines ,
existing both in soluble form and as a transmembrane form , cxcl16 possessed functions seemingly more nuanced than other well - described chemokines . in soluble form
it induces immunocyte chemotaxis , while in transmembrane form cxcl16 mediates cell - cell adhesion [ 1 , 3 , 4 ] . previous findings from in vitro studies using exogenous scxcl16 suggested that scxcl16 promotes cell proliferation and invasion . although matsushita et al . associated high preoperative levels of scxcl16 with liver recurrence and poor prognosis in colorectal cancer patients , little
was known about the functions of tm - cxcl16 except that high expression of cxcl16 in cancer tissues correlated with favorable prognosis in renal and colorectal cancer patients [ 12 , 13 ] .
these observed differences between tm - cxcl16 and scxcl16 indicate a complicated function for cxcl16 in cancer .
we therefore sought to identify a specific function for tm - cxcl16 through in vivo and in vitro breast cancer models . in this study , we show for the first time that upregulation of cxcl16 suppresses migration and invasiveness of breast cancer cells in vitro and delays progression of tumor growth in vivo .
these results reveal a protective function of cxcl16 in breast carcinogenesis and present valuable clues to better understanding of the mechanisms of breast cancer progression .
our measurements of both protein and mrna levels of cxcl16 reveal high expression in mcf-7 cell line , which are positive for estrogen receptor ( er ) and progesterone receptor ( pr ) and therefore considered less aggressive ; levels were lower in the receptor triple - negative cancer cell line of mda - mb-231 which is considered more highly aggressive .
this inverse correlation between cxcl16 expression and migration or invasion suggested that cxcl16 play another role as tumor suppressor in inhibiting the migration and invasiveness of breast cancer cells .
as discussed above , tm - cxcl16 plays a pivotal role as a tumor suppressor on the development of breast cancer cells ; that is , the high expression of cxcl16 or overexpression of cxcl16 significantly suppressed the invasion and migration and induced apoptosis of breast cancer cells ; in contrast , low expression of cxcl16 or downregulation of cxcl16 promoted the invasion and migration of breast cancer cells .
supportively , the studies have reported that the reduced cell - cell adhesiveness is considered one morphological hallmark of malignant tumors , and homotypic adhesion was described to reduce the invasive potential of tumor cells .
consistent with previous reports , we found that cxcl16 and cxcr6 were coexpressed in breast cancer cells but to inverse extent .
when expressed as transmembrane protein , cxcl16 could directly promote cell - cell adhesion by combining with cxcr6 on cell surface .
thus , we speculate here that transmembrane cxcl16 facilitates the cell - cell adhesion like other well described cell adhesion molecules , such as e - cadherin , which subsequently prevent the detachment of individual tumor cells from the tumor aggregation and ultimately inhibit cell migration and invasion .
heparin - like glycosaminoglycans are long unbranched polysaccharides consisting of a repeating disaccharide unit that expressed on proteoglycan components of cell surface and extracellular matrix .
cell surface glycosaminoglycans could bind positively charged regions on the protein and lead to enhanced local concentrations of chemokines . for example , heparin binds to the strong positive potential on cxcl12 and stabilizes the cxcl12 dimer on cell surface .
moreover , the presence of cell surface gags could enhance the activity of chemokines including mip-1a , rantes , or mip-1b by sequestrating them onto the cell surface .
there also has been evidence that gag is important for cxcl16 recognition and that cxcl16 was able to react with heparin microarrays containing oligosaccharides .
however , it remains unknown what the effect of gag - cxcl16 interactions is and how heparin - like glycosaminoglycan affects the activity of cxcl16 .
another significant finding was that overexpression of cxc16 enhanced apoptosis in mda - mb-231 in conjunction with upregulation of caspase-3 , a crucial enzyme in endogenous apoptosis , suggesting cxcl16 promotes caspase-3-dependent apoptosis .
aberrant balance between proliferation and apoptosis is clearly implicated as one major characteristic in tumor development . however , in this study the apoptosis index or caspase-3 activity in mcf-7 cells is not determined , because the mcf-7 cell line was considered not to express the enzyme caspase-3 .
we discovered no role for cxcl16 in the proliferation of breast cancer cell lines in our study , unlike other tumor suppressor studies .
it has been speculated that tumor cell lines are normally transformed and have multiple genetic alterations , which may contribute to the noneffect of cxcl16 on the breast cancer proliferation .
egfr overexpression is well - studied in breast cancer , and egfr - driven signaling pathways such as pi3k / akt / mtor , jak / stat , and ras / raf / mapk were associated with cell proliferation and survival . since
egfr overexpression is common in breast cancer , the impact of cxcl16 alteration on proliferation may be masked by egfr overexpression or other genetic alterations .
these are speculative explanations for how cxcl16 appears not to affect proliferation of breast cancer cells .
in addition , since cxcl16 exists both as a transmembrane form and a soluble form , and the former could be cleavaged off into the soluble form , it is difficult to distinguish precisely which form is being studied in which cancer experiments . in fact , it has been reported that a molecule adam10 mediates the conversion of transmembrane cxcl16 into soluble cxcl16 dynamically [ 4 , 5 ] .
therefore , in this study we defined cxcl16 as  cellular cxcl16  , which exists predominantly in the cell membrane . to characterize it , we employed flow cytometry to quantify cxcl16 localized on cell surfaces in living cancer cells .
we report high cell surface expression of cxcl16 on living cells , in proportion to total cxcl16 expression by cells .
our data suggest that the transmembrane cxcl16 is involved in breast cancer cells with important clinical significance . in summary ,
the current study demonstrated that cellular cxcl16 expression was negatively correlated with the migration and invasion of breast cancer cells .
 
 background . 
 increasing evidence argues that soluble cxcl16 promotes proliferation , migration , and invasion of cancer cells in vitro . 
 however , the role of transmembrane or cellular cxcl16 in cancer remains relatively unknown . in this study 
 , we determine the function of cellular cxcl16 as tumor suppressor in breast cancer cells . 
 methods . 
 expression of cellular cxcl16 in breast cancer cell lines was determined at both rna and protein levels . in vitro and in vivo studies that overexpressed or downregulated cxcl16 were conducted in breast cancer cells . results . 
 we report differential expression of cellular cxcl16 in breast cancer cell lines that was negatively correlated with cell invasiveness and migration . 
 overexpression of cxcl16 in mda - mb-231 cells led to a decrease in cell invasion and migration and induced apoptosis of the cells ; downregulation of cxcl16 in mcf-7 cells increased cell migration and invasiveness . 
 consistent with the in vitro data , cxcl16 overexpression inhibited tumorigenesis in vivo 
 . conclusions . 
 cellular cxcl16 suppresses invasion and metastasis of breast cancer cells in vitro and inhibits tumorigenesis in vivo . 
 targeting of cellular cxcl16 expression is a potential therapeutic strategy for breast cancer . 