Patent Application: US-201213979615-A

Abstract:
the present invention relates to a novel molecular marker for pancreatic cancer stem cells and pancreatic cancer , to a marker detection method , and to a screening method . the present invention is a marker discovered from the cell lines of pancreatic cancer , wherein the marker may detect pancreatic cancer , in particular early pancreatic cancer , through the detection of a pancreatic cancer stem cell marker . in addition , the marker of the present invention may enable an accurate diagnosis and prognosis analysis of pancreatic cancer .

Description:
the present invention will now be described in further detail by examples . it would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples . unless otherwise described in the specification , solid / solid , solid / liquid and liquid / liquid indicate ( weight / weight ) parts by weight or %, ( weight / volume ) parts by weight or %, and ( volume / volume ) parts by weight or %, respectively . isolation and culture of sphere having properties of cancer stem cells from human pancreatic cancer cell lines eight pancreatic cancer cell lines ( hpac , aspc1 , bxpc3 , capan1 , capan2 , cfpac , miapaca2 , and panc 1 ) were purchased from the american type culture collection ( atcc , manassas , va ., us ) and cultured in media according to the protocol of atcc instructions . i . e ., aspc - 1 and bxpc - 3 cell lines were cultured in rpmi1640 ( invitrogen gibco , grand island , n . y .) supplemented with 10 % fetal bovine serum ( fbs , hyclone , logan , utah , us ). capan - 1 and cfpac - 1 cell lines were cultured in imdm ( invitrogen gibco ) supplemented with 10 % fbs . capan - 2 cell line was cultured in mccoy &# 39 ; s 5a ( invitrogen gibco ) supplemented with 10 % fbs . mia paca - 2 cell line was cultured in dmem ( invitrogen gibco ) supplemented with 10 % fbs and 2 . 5 % horse serum ( hyclone ). hpac cell line was cultured in dmem / ham &# 39 ; s f12 ( d / f12 ; invitrogen gibco ) supplemented with 10 % fbs . panc - 1 cell line was cultured in dmem supplemented with 10 % fbs . for sphere formation , pancreatic cancer cell lines were plated 1000 cell / ml in serum - free dmem / f12 supplemented with egf ( 10 ng / ml , r & amp ; d systems inc ., minneapolis , minn . ), bfgf ( 10 ng / μl , r & amp ; d systems inc . ), 1 its ( insulin transferring selenium , gibco ) and 0 . 5 % fbs on corning costar ultra - low attached 6 - well culture dishes ( corning inc ., corning , n . y ., us ), and cultured for 7 - 10 days . the adherent cells cultured with the same media by attaching on culture plates ( nalgene nunc int ., rochester , n . y .) were used as a control group . total rna was extracted with rneasy mini prep kit ( qiagen , valencia , calif ., us ). each 2 μl of rna was reverse - transcribed using superscript ii ( gibco brl , grand island , n . y .) at 42 ° c . for 1 hour . expressions of cancer stem cell - related genes were analyzed by pcr using primers and taq dna polymerase ( takara biochemicals , tokyo , japan ). expression of beta - actin gene was used as a control . the primers , pcr temperature and cycles are described in table 1 . expression analysis of cancer stem cell markers in sphere cells through fluorescence - activated cell sorting after incubations of adhesive hpac cells and hpac - spheres , cells were treated with accutase ( sigma - aldrich , st . louis , mo .) to prepare suspensions with the same number of single cells . afterward , adhesive cells and spheres were stained with cancer stem cell marker antibodies as follows : fitc - cd44 ( bd biosciences pharmingen , san diego , calif . ), pe - prom2 ( bd biosciences pharmingen , san diego , calif . ), pe - epha2 ( bd biosciences pharmingen , san diego , calif . ), pe - cd130 ( bd biosciences pharmingen , san diego , calif . ), pe - cd271 ( bd biosciences pharmingen , san diego , calif . ), pe - cd24 ( bd biosciences pharmingen , san diego , calif . ), pe - cd133 ( bd biosciences pharmingen , san diego , calif .) and pe - cd117 ( bd biosciences pharmingen , san diego , calif .). fluorescence - activated cell sorting was carried out using bd lsr ii ( bd biosciences , franklin lakes , n . j .) and analyzed by bd facsdiva software . well - known stem cell signaling inhibitors dapt ( calbiochem , san diego , calif ., usa ), cyclopamine - kaad ( calbiochem , san diego , calif ., usa ), licl ( sigma - aldrich , inc ., st . louis , mo . ), atra ( sigma - aldrich , inc ., st . louis , mo . ), ov ( sigma - aldrich , inc ., st . louis , mo .) were added added into sphere culture medium , and cultured . after 7 days , sphere formation was observed . adhesive hpac cells and hpac - spheres were treated with 0 . 25 % trypsin edta ( gibco brl , grand island , n . y .) to prepare suspensions with the same number of single cells . afterward , they were directly in situ - transplanted into pancreas of nod / scid mice ( charles river , japan ) and observed to tumorigenicity of hpac cells and hpac - spheres . in order to analyze cancer stem cell - related secretory protein in media of sphere cells , hpac cells and capan 1 cells were prepared to spheres or adherent cells for 7 - 10 days . then , their media were changed to serum - free dmem / f12 ( gibco brl , grand island , n . y .) and they were incubated 24 hours . the culture media supernatants of spheres and adherent cells were collected using centrifugation and filter . the supernatants containing various secretory proteins were concentrated using amicon ultra centifugal filter device with 3 kda molecular - mass cut off ( mwco ), and carried out to proteomics analysis ( 2 - d gel analysis and maldi - tof ). spots showing differences after stained gel image analysis were cutted off , and identified through maldi - tof ( matrix assisted laser desorption / ionization - time of flight ) analysis . mascot was used as the database search engine ( na k et al ., proteomics ( 9 ): 3989 - 3999 , 2009 ). expression analysis of pancreatic cancer stem cell - related secretory proteins in sphere cell media through western blot in order to verify expressions of secretory proteins identified by proteomics analysis in the media of adhesive hpac cells , hpac - spheres , adhesive capan 1 cells and capan 1 - spheres , the media supernatants were collected using centrifugation and filter . the supernatants were concentrated using amicon ultra centifugal filter device with 3 kda molecular - mass cut off ( mwco ) to quantify proteins . each of 25 μg of the quantified proteins was loaded into 10 % sds - polyacrylamide gel and electrophorezed . the proteins fractionized by size using the electrophoresis were transferred onto pvdf membranes ( millipore corporation , billerica , mass ., usa ). to reduce nonspecific binding , the membranes were blocked for 1 hour in tbs - t buffer ( tris - buffered saline / 0 . 05 % tween - 20 ) containing 5 % non - fat milk . then , each membrane was incubated with the primary antibody corresponding to secretory proteins identified by proteomics analysis at 4 ° c . for overnight . the primary antibodies were mouse monoclonal akr1b1 ( santa cruz biotechnology inc , santa cruz , ca ), rabbit polyclonal hsp90 ( santa cruz biotechnology inc , santa cruz , ca ), mouse monoclonal aldh ( bd biosciences pharmingen , san diego , calif . ), mouse monoclonal vimentin ( santa cruz biotechnology inc , santa cruz , ca ) and mouse monoclonal glrx3 ( abnova corporation , taipei ). the membrane was washed with tbs - t buffer , and incubated with hrp ( horseradish peroxidase )- conjugated secondary antibody ( santa cruz biotechnology inc , santa cruz , ca ) for 1 hour . each protein was detected using enhanced chemiluminescence system ( pierce , rockford , ill .). verification on expressions of mrna and protein of glrx3 in pancreatic cancer cell lines to verify glrx3 expression , mrna and protein were extracted in eight pancreatic cancer cell lines ( aspc - 1 , bxpc - 3 , capan - 1 , capan - 2 , cfpac - 1 , hpac , panc - 1 and miapaca - 2 ) and hpde ( human pancreatic ductal epithelial cell ). total rna was extracted with rneasy mini prep kit ( qiagen , valencia , calif ., us ), and reverse - transcribed using superscript ii ( gibco brl , grand island , n . y .). expressions of glrx3 gene in pancreatic cancer cell lines were analyzed by pcr using glrx3 primers and taq polymerase . expression of beta - actin gene was used as a control . expression analysis of glrx3 protein in pancreatic cancer cell lines was performed in total proteins and secretory proteins . total proteins were extracted using cell lysis buffer ( 50 mm tris , 150 mm nacl , 25 mm β - glycerophosphate , 25 mm naf , 0 . 5 m edta , 1 % np - 40 , 0 . 1 mm pmsf , 1 % protease inhibitors cocktail ( roche )). secretory proteins were prepared as follows : culture media cultured under serum - free condition were centrifuged to remove cell debris , and precipitated by ice - cold acetone in the final volume of 80 %. the precipitated secretory proteins were washed with 100 % ice - cold acetone , dried by centrifugal vacuum dryer , and dissolved in cell lysis buffer . total proteins and secretory proteins were quantified . each of 25 ng of the quantified proteins was loaded into 10 % sds - polyacrylamide gel and electrophorezed . the proteins fractionized by size using the electrophoresis were transferred onto pvdf membrane ( millipore corporation , billerica , mass ., usa ). to reduce nonspecific binding , the membrane was blocked for 1 hour in tbs - t buffer ( tris - buffered saline / 0 . 05 % tween - 20 ) containing 5 % non - fat milk . then , the membrane was incubated with the primary antibody to glrx3 ( abnova corporation , taipei , china ) at 4 ° c . for overnight . the membrane was washed with tbs - t buffer , and incubated with hrp ( horseradish peroxidase )- conjugated secondary antibody ( santa cruz biotechnology inc , santa cruz , ca , usa ) for 1 hour . the protein was detected using west pico chemiluminescent substrate system ( pierce , rockford , ill .). verification on over - expression of glrx3 in the cell fraction having properties of pancreatic cancer stem cells to fractionize cells having properties of cancer stem cells , hpac and cfpac - 1 cell lines were treated with accutase ( sigma - aldrich , st . louis , mo .) to prepare suspensions with the same number of single cells . afterward , the cells were stained on ice for 10 min with cancer stem cell marker as follows : pe - cd24 ( bd biosciences pharmingen , san diego , calif . ), apc - cd44 ( bd biosciences pharmingen , san diego , calif .) and fitc - esa ( bd biosciences pharmingen , san diego , calif .). in addition , the cells were stained with purified mouse igg1 , λ - fitc , igg2a , κ - pe and igg2b , κ - apc ( bd biosciences pharmingen , san diego , calif ., usa ) as isotype controls to antibodies . fluorescence - activated cell sorting was carried out using facsaria ii ( bd immunocytochemistry system , franklin lakes , n . j ., us ). each total rna of fractions in cells expressing 3 types of markers ( cd24 +/ cd44 +/ esa +) and cells non - expressing 3 types of markers ( cd24 −/ cd44 −/ esa −) was extracted with rneasy mini prep kit ( qiagen , valencia , calif . ), and reverse - transcribed using superscript ii ( gibco brl , grand island , n . y ., us ). expressions of glrx3 gene were analyzed by pcr using glrx3 primers and taq polymerase . expression of beta - actin gene was used as a control . to inhibit glrx3 , sirna ( invitrogen stealth ™ sirna duplex oligoribonucleotides ) to glrx3 was used . sense sirna sequence was uga ggg agu ucu uua gcu aac ucu g , and antisense sirna sequence was cag agu uag cua aag aac ucc cuc a . med gc ( invitrogen ) was used as negative control sirna . cfapc - 1 cells were transfected with glrx3 or negative control sirna using rnaimax ( invtirogen , carlsbad , calif ., us ), and incubated for 72 hours . the cells were treated with accutase ( sigma - aldrich , st . louis , mo .) to prepare suspensions with the same number of single cells . afterward , the cells were stained with pe - cd24 ( bd biosciences pharmingen , san diego , calif . ), apc - cd44 ( bd biosciences pharmingen , san diego , calif .) and fitc - esa ( bd biosciences pharmingen , san diego , calif .). fluorescence - activated cell sorting was carried out using bd lsr ii ( bd biosciences , franklin lakes , n . j .) and analyzed by bd facsdiva software . to verify inhibitory effects of glrx3 expression , shrna ( sabiosciences sure silencing ™ shrna plasmid ) to glrx3 and control shrna were prepared . hpac cell line was transfected with the shrnas to establish glrx3 knock - down ( k / d ) cell line . expressions of glrx3 mrna and protein were investigated . shrna sequence to glrx3 was gtg gaa att ctt cac aaa cat , and control shrna sequence was gga atc tca ttc gat gca tac . selection marker was puromycin ( 2 . 0 ng / ml ). at one day before gene transfection , an equal number of cells were attached on the plates . then , shrna to glrx3 and control shrna were mixed with lipofectamine - 2000 ( invitrogen , carlsbad , calif ., usa ) to transfect the cells . at 24 hours after gene transfection , the media were changed to media containing 2 ng / ml of puromycin , and maintained for 3 days . survival cells in media containing puromycin were plated 1 cell / well on 96 - well plate , and cultured . mrna and protein of the cells proliferated in each well were extracted , and glrx3 expression changes were investigated by pcr and western blot , whereby two clones of the glrx3 k / d cell line and two clones of the control were finally obtained . in order to verify cancer - related characteristic changes of the obtained glrx3 k / d , doubling time assay and wound - healing assay were carried out . for cell doubling times , 1 . 25 × 10 4 cells were seeded into 24 - well plate . the cells were detached by 0 . 25 % trypsin - edta and counted every a regular hour for 5 days using hematocytometer . for wound - healing assay , the cells were grown until nearly 100 % confluent , and linear wound was created in the confluent monolayer using pipette tip . wound healing ability was observed according to time under a microscope , and photographed ( olympus dp50 ). analysis of cancer stem cell - related characteristic changes in glrx3 knock - down cell line as glrx3 were revealed as cancer stem cell - related protein , cancer stem cell - related characteristic changes induced by the glrx3 k / d were investigated . first , ability to form spheres of the glrx3 k / d cell line and the control cell line was verified . to achieve this , two clones of the glrx3 k / d cell line and two clones of the control were sphere - cultured for 7 days . for clonogenic assay , 0 . 6 % agar in cell media was boiled , hardened on 24 - well plate ( lower layer media ). the cells at density of 1000 cell / well and 0 . 3 % agar - cell media ( upper layer media ) were mixed , and hardened on lower layer media . once 0 . 3 % upper layer media was hardened , 500 μl / well of media was added on the upper layer media . adding the media every third day , the cells were cultured for 3 - 4 weeks . afterward , the media on agar was removed , stained with crystal violet to count the number of colony . expression analysis of pancreatic cancer stem cell - related secretory proteins in human pancreatic cancer tissue through immunohistochemistry pancreatic tissue slides were deparaffinized in xylene and rehydrated in a graded ethanol series . endogenous peroxidase was blocked by immersing the slide in 0 . 3 % hydrogen peroxide in methanol at room temperature for 20 minutes . microwave antigen retrieval was performed in citrate buffer ( 0 . 01m , ph 6 . 0 ) for 4 min . the slides were blocked by soaking in 10 % normal donkey serum in pbs for 1 hour to remove non - specific background . then , the slides were incubated with primary antibodies in 1 : 200 dilution at 4 ° c . for overnight . the primary antibodies were mouse monoclonal agpat4 ( abnova corporation , taipei ), mouse monoclonal akr1b1 ( santa cruz biotechnology inc , santa cruz , ca ), rabbit polyclonal hsp90 ( santa cruz biotechnology inc , santa cruz , ca ), mouse monoclonal aldh ( bd biosciences pharmingen , san diego , calif . ), rabbit polyclonal msk2 ( santa cruz biotechnology inc , santa cruz , ca ), mouse monoclonal vimenti n ( santa cruz biotechnology inc , santa cruz , ca ) and mouse monoclonal glrx3 ( abnova corporation , taipei ). subsequent reactions were carried out using envision kit ( dakocytomation , carpineteria , calif ., usa ) according to the manufacturer &# 39 ; s protocols . finally , the slides were incubated with 3 , 3 ′- diaminobenzidine ( dakocytomation , carpinteria , calif ., usa ), and counterstained with harris hematoxylin ( sigma - aldrich , inc ., st . louis , mo .). the results were classified as − ( negative ), + ( light brown ), ++ ( middle brown ) and +++ ( strong brown ) according to staining intensity . expression analysis of pancreatic cancer stem cell - related secretory proteins in sera of human normal , chronic pancreatitis and pancreatic cancer patients through western blot in sera of human normal , chronic pancreatitis and pancreatic cancer patients , expressions of pancreatic cancer stem cell - related secretory proteins were analyzed through western blot . to verify expressions of pancreatic cancer stem cell - related secretory proteins developed in sera of human normal , chronic pancreatitis and pancreatic cancer patients , normal serum 5 cases , chronic pancreatitis serum 5 cases and pancreatic cancer serum 20 cases were selected . after removals of 6 principal proteins ( albumin , transferrin , igg , iga , haptoglobin and anti - trypsin ) in total 30 cases of the sera using mars ( multiple affinity removal column system ), the sera were concentrated using amicon ultra centifugal filter device with 3 kda molecular - mass cut off ( mwco ) to quantify proteins . each of 40 μg of the quantified proteins was loaded into 10 % sds - polyacrylamide gel and electrophorezed . the proteins fractionized by size using the electrophoresis were transferred onto pvdf membranes ( millipore corporation , billerica , mass ., usa ). to reduce nonspecific binding , the membranes were blocked for 1 hour in tbs - t buffer ( tris - buffered saline / 0 . 05 % tween - 20 ) containing 5 % non - fat milk . then , each membrane was incubated with the primary antibody ( 1 : 500 dilution ) corresponding to proteins at 4 ° c . for overnight . the primary antibodies were rabbit polyclonal msk2 ( santa cruz biotechnology inc , santa cruz , ca ), mouse monoclonal vimentin ( santa cruz biotechnology inc , santa cruz , ca ), mouse monoclonal aldh ( bd biosciences pharmingen , san diego , calif .) and mouse monoclonal glrx3 ( abnova corporation , taipei ). the membrane was washed with tbs - t buffer , and incubated with hrp ( horseradish peroxidase )- conjugated secondary antibody ( santa cruz biotechnology inc , santa cruz , ca ) for 1 hour . each protein was detected using enhanced chemiluminescence system ( pierce , rockford , ill .). in order to verify expressions of glrx3 in clinical patients ( normal control patients 28 cases , chronic pancreatitis patients 9 cases and pancreatic cancer patients 57 cases ), elisa was performed using elisa kit according to the manufacturer &# 39 ; s protocols . the serum was diluted in pbs to 1 : 10 ratio to use . the standard concentrations were prepared by means of ½ serial dilution from 10 nm / ml . the diluted patient serum and standard concentrations were added into 96 - well of glrx3 elisa kit , respectively , and incubated for 2 hours at 37 ° c . 100 μl , of detection reagent a was added into each well , and incubated for 1 hour at 37 ° c . after removals of all solutions , the wells were washed 3 times with washing buffer . 100 μl , of detection reagent b was added into each well , and incubated for 30 min at 37 ° c . after removal of reagent , the wells were washed 5 times , added with 90 μl of substrate solution , incubated at 37 ° c . for 15 - 20 min , added with 50 μl of stop solution , and measured the absorbance at 450 nm . isolation and culture of sphere having properties of cancer stem cells from human pancreatic cancer cell lines eight human pancreatic cancer cell lines were tested for the sphere formation which is property of cancer stem cell . as a result , three cancer cell lines including hpac , capan 1 and capan 2 showed the ability to form the sphere . among the sphere forming cell lines , hpac and capan 1 were further analyzed ( fig1 ). it was determined that hpac - spheres showed greater activity of stem cell - related signaling pathways such as notch , hedgehog , and wnt than that of adhesive hpac cells . early embryonic development related genes such as oct4 , nanog and stat3 which represent multipotency of stem cell were over - expressed . in addition , cancer stem cell markers abcg2 , pten were over - expressed ( fig2 a - b ). expression analysis of cancer stem cell markers in sphere cells through fluorescence - activated cell sorting in fluorescence - activated cell sorting , it was determined that well - known cancer stem cell markers cd44 , prom2 , epha2 , cd130 and cd271 were over - expressed in adhesive hpac cells and hpac - spheres ( fig3 ). it was determined that ability to form sphere in hpac - spheres was completely inhibited or reduced by well - known stem cell signaling inhibitors ( fig4 a - c ; dapt is an inhibitor to notch ; kaad is an inhibitor to hh ; licl is an inhibitor to gsk3b ; atra is an inhibitor to oct4 ; and ov is an inhibitor to nanog ). hpac - spheres were in situ - implanted into pancreas of nod / scid mouse to validate tumorigenicity . the hpac - spheres injected group was analyzed to be significantly higher than that for the adhesive hpac cells injected group . in addition , pulmonary metastasis and peritoneal metastasis were observed in the hpac - spheres injected group ( fig5 a - c ). hpac - spheres express cancer stem cell - related genes and markers . it would be understood that pancreatic cancer stem cell - specific markers may be predicted by investigating profiles of proteins secreted from hpac - spheres , based on these results showing enhanced tumorigenic potential ( fig1 - 5 c ). 587 paired spots were detected in hpac - spheres , adhesive hpac cells , capan 1 - spheres and adhesive capan 1 cells . among them , 55 spots which were increased more than two times in hpac - spheres as compared to adhesive hpac cells were detected . 41 spots in the 55 spots were subject to maldi - tof to identify pancreatic cancer stem cell - related secretory protein ( table 2 ). proteins including hsp90ab1 , aldh and vimentin which have been reported to be associated with cancer stem cell were identified . these results suggest that the secretory proteins described in table 1 have potential to be pancreatic cancer stem cell - related secretory proteins . expression verification of pancreatic cancer stem cell - related secretory proteins in sphere cell media through western blot in order to verify expressions of pancreatic cancer stem cell - related secretory proteins identified by maldi - tof analysis , the media of adhesive hpac cells , hpac - spheres , adhesive capan 1 cells and capan 1 - spheres were subject to western blot . it was verified that 5 secretory proteins including glrx3 were increased in hpac - spheres and capan 1 - spheres . verification on expressions of mrna and protein of glrx3 in pancreatic cancer cell lines to verify glrx3 expression , mrna and protein were extracted in eight pancreatic cancer cell lines ( aspc - 1 , bxpc - 3 , capan - 1 , capan - 2 , cfpac - 1 , hpac , panc - 1 and miapaca - 2 ) and hpde ( human pancreatic ductal epithelial cell ). as a result , it was determined that both of mrna and protein of glrx3 were over - expressed in eight pancreatic cancer cell lines . in addition , it was found that pancreatic cancer cell lines except for hpac extracellularly secreted glrx3 protein through western blot in the media ( fig7 ). verification on over - expression of glrx3 in the cell fraction having properties of pancreatic cancer stem cells hpac was fluorescence - stained by cancer stem cell marker cd24 , cd44 and esa . then , cells expressing 3 types of markers ( cd24 +/ cd44 +/ esa +) and cells non - expressing 3 types of markers ( cd24 −/ cd44 −/ esa −) were fractionized . as a result , it was verified that glrx3 in cells expressing 3 types of markers ( cd24 +/ cd44 +/ esa +) were over - expressed than that of cells non - expressing 3 types of markers ( cd24 −/ cd44 −/ esa −). the same results were shown in cfpac - 1 ( fig8 ). in addition , the fractions of cancer stem cells were changed by inhibition of glrx3 expression . it was verified that fraction of cd24 +/ cd44 +/ esa + was decreased to 29 . 3 % from 70 . 4 % where sirna to glrx3 were treated ( fig9 ). to verify inhibitory effects of glrx3 expression , shrna to glrx3 and control shrna were prepared . hpac cell line was transfected with the shrnas to establish glrx3 knock - down ( k / d ) cell line . expressions of mrna and protein of glrx3 in the established glrx3 knock - down ( k / d ) cell line were significantly reduced ( fig1 a ). in order to verify cancer - related characteristic changes of the glrx3 k / d , two clones of the glrx3 k / d cell line and two clones of the control were selected . it was determined that the cell proliferation rate in the glrx3 k / d cell line was decreased as compared to the control ( fig1 b ), and a result of wound - healing assay , cell migration activity in the glrx3 k / d cell line was decreased as compared to the control ( fig1 c ). analysis of cancer stem cell - related characteristic changes in glrx3 knock - down cell line as glrx3 were revealed as cancer stem cell - related protein , cancer stem cell - related characteristic changes induced by the glrx3 k / d were investigated . first , ability to form spheres of the glrx3 k / d cell line and the control cell line was verified ( fig1 a ). as a result , the spheres were not formed in the glrx3 k / d cell line , whereas the spheres were formed in the control cell line . these results are shown as the same results in the sphere culture that glrx3 expression was increased . in addition , as a result of clonogenic assay showing viability and proliferation , the glrx3 k / d cell line formed fewer colonies than the control cell line ( fig1 b ). in these results , glrx3 showed not only possibility as oncogenic markers related to pancreatic cancer stem cells , but also as targets for pancreatic cancer therapeutic agents based on inhibitory effects to various cancer - related characteristic induced by glrx3 inhibition . expression verification of pancreatic cancer stem cell - related secretory proteins in human pancreatic cancer tissue agpat4 was expressed in tma slides of total 29 cases , and classified as “++” ( 15 cases ) and “+++” ( 14 cases ) in 100 % of pancreatic cancer patient tissues ( panel ( a ) in fig1 ). akr1b1 was expressed in tma slides of total 27 cases , and classified as “−” ( 10 cases ), “+” ( 15 cases ) and “++” ( 2 cases ) in 62 . 9 % of pancreatic cancer patient tissues ( panel ( b ) in fig1 ). hsp90 was expressed in tma slides of total 28 cases , and classified as “+” ( 1 case ), “++” ( 19 cases ) and “+++” ( 8 cases ) in 100 % of pancreatic cancer patient tissues ( panel ( c ) in fig1 ). aldh was expressed in tma slides of total 26 cases , and classified as “−” ( 4 cases ), “+” ( 8 cases ), “++” ( 7 cases ) and “+++” ( 7 cases ) in 84 . 6 % of pancreatic cancer patient tissues ( panel ( d ) in fig1 ). msk2 was expressed in tma slides of total 27 cases , and classified as “−” ( 1 case ), “+” ( 5 cases ), “++” ( 8 cases ) and “+++” ( 13 cases ) in 96 . 2 % of pancreatic cancer patient tissues ( panel ( e ) in fig1 ). vimentin was mainly stained in extracellular interstitium . although a lot of the stained cells formed tubular structure , they were thought to be non - cancer cells . vimentin was hardly stained in cytoplasm of cancer cells . vimentin was focal - stained in 1 case among 28 cases of pancreatic cancer tissues ( panel ( f ) in fig1 ). glrx3 was expressed in tma slides of total 25 cases , and classified as “−” ( 11 cases ), “+” ( 11 cases ) and “++” ( 4 cases ) in 57 . 6 % of pancreatic cancer patient tissues ( panel ( g ) in fig1 ). paraffin sections in surgical tissue of pancreatic cancer patients were stained by immunohistochemistry to verify expressions of pancreatic cancer stem cell - related secretory proteins . it could be appreciated that all 7 pancreatic cancer stem cell - related secretory proteins including glrx3 ( developed by the present invention ), hsp90ab1 , aldh and vimentin which have been already reported to be associated with cancer stem cell were strongly expressed in pancreatic cancer tissue as compared to normal pancreatic tissue ( panel ( a )-( g ) in fig1 ). expression verification of secretory proteins developed in sera of human normal , chronic pancreatitis and pancreatic cancer patients as the absence of commercial elisa kit for the developed secretory proteins , some proteins were subject to western blot to verify expressions . to verify expressions of pancreatic cancer stem cell - related secretory proteins developed in sera of human normal , chronic pancreatitis and pancreatic cancer patients , normal serum 5 cases , chronic pancreatitis serum 5 cases and pancreatic cancer serum 20 cases were selected . to compare pancreatic cancer stem cell - related secretory proteins developed in the present invention with conventional pancreatic cancer markers ( ca 19 - 9 and cea ), levels of ca 19 - 9 and cea in serum of the selected pancreatic cancer patients ( pc2 - pc10 ) were shown in fig1 a - b . prior to verification of the developed secretory protein expressions through western blot , 6 principal proteins ( albumin , transferrin , igg , iga , haptoglobin and anti - trypsin ) in total 30 cases of the sera were removed using mars ( multiple affinity removal column system ), and then , the sera were concentrated using amicon ultra centifugal filter device with 3 kda molecular - mass cut off ( mwco ) to quantify proteins . an equal concentration of the proteins was used in western blot . secretory proteins vimentin and aldh which have been well - known to be associated with cancer stem cells showed results that they were over - expressed in pancreatic cancer patient serum as compared to normal and chronic pancreatitis patient sera . in addition , whereas glrx3 as the developed pancreatic cancer stem cell secretory protein was very low - expressed in normal serum , glrx3 was high - expressed in more than 50 % of pancreatic cancer patient serum as compared to normal and chronic pancreatitis patient sera ( fig1 a - 18 b ). in order to verify expressions of glrx3 in clinical patients , elisa was performed using elisa kit ( uscn glrx3 elisa kit ). as a result of elisa in normal control 28 cases , chronic pancreatitis patients 9 cases and pancreatic cancer patients 57 cases , it was determined that glrx3 was high - expressed in pancreatic cancer patient blood ( fig1 a ). particularly , there were differences of survival periods according to glrx3 level that survival periods of patients showing less than 1800 ng / ml of glrx3 level were 463 days , whereas survival periods of patients showing more than 1800 ng / ml of glrx3 level were 155 days ( fig1 b ). cancer stem cells are cancer cells that possess the abilities to maintain and regenerate cancer tissues like normal stem cells . cancer stem cells are not only thought to be involved in oncogenesis , but also have been reported that they profoundly influence cancer recurrence , metastasis or resistance to chemotherapy by involving regeneration of cancer cells after anticancer therapies ( bb zhou et al . nat rev drug discov ., 8 : 806 - 823 ( 2009 )). cancer stem cells are a rare population of heterogenous cancer cells and they have been known as tumor initiating cells which have ability to form tumor . where these cells are suspension - cultured in vitro , they form spheres . it has been reported that the cells obtained from sphere - culture have stronger ability to form tumor than that of the original cell line ( a . dubrovska et al . proc natl acad sci usa ., 106 : 268 - 273 ( 2009 )). in addition , genes and proteins related to several cancers and cancer stem cell have been known to be strongly expressed in these cells than that of the original cell line . in the present invention , it was verified that spheres formed from pancreatic cancer cell lines have the capacity to self - renewal by generating sub - spheres , and they have aberrant activation of stem cell - related signaling pathways such as notch , hedgehog , and wnt ( t . reya et al . nature , 414 : 105 - 111 ( 2001 )). in addition , it could be determined that early embryonic development related genes such as oct4 , nanog and stat3 which represent multipotency of stem cell were over - expressed . besides , pten which plays a pivotal rule in cancer survival and maintenance were over - expressed , of which showed the results consistent with previous reports related to stem cells ( j . zhou et al . proc natl acad sci usa ., 104 : 16158 - 16163 ( 2007 )). as a result of fluorescence - activated cell sorting , it could be determined that well - known cancer stem cell markers cd44 , prom2 , epha2 , cd130 and cd271 were over - expressed in adhesive hpac cells and hpac - spheres . based on these results , the present inventors have carried out proteomics analysis in order to connect roles of cancer stem cells recognized as the early symptoms of oncogenesis , and to identify and verify proteins secreted in sphere - culture , whereby proteins specifically secreted in pancreatic cancer patients were validated . as a result of proteomics , the secretory proteins hsp90 , vimentin , hsp27 , aldh , agpat4 , akr1b1 , msk2 and glrx3 were identified . in pancreatic cancer tissues , these secretory proteins expressions were significantly increased as compared to normal tissues . moreover , in pancreatic cancer patient sera , these secretory proteins expressions were significantly increased as compared to normal tissues . although proteins were subject to western blot to verify expressions in serum due to the absence of commercial elisa kit for the developed secretory proteins , it could be validated that expressions of the present secretory proteins were increased in pancreatic cancer patient sera . particularly , glrx3 expression was increased in pancreatic cancer patient serum , and survival period of pancreatic cancer patients with high - expression of glrx3 was relatively lower than that of pancreatic cancer patients with low - expression of glrx3 . in addition , where glrx3 expression was inhibited , various cancer - and caner stem cell - related characteristics were decreased . based on these results , glrx3 were validated that it may be used to targets for diagnosing or treating as well as markers for prognosing pancreatic cancer . in addition , many proteins were together identified as follows : hsp90 reported as therapeutic target for various cancers ( l . whitesell and s . l . lindquist , nat rev cancer , 5 : 761 - 772 ( 2005 )); vimentin as a marker for epithelial to mesenchymal transition associated with cancer stem cells ( j m . lee et al . j cell biol ., 172 : 973 - 981 ( 2006 ); pb . gupta et al . cell ., 138 : 645 - 659 ( 2009 )); hsp27 associated with chemotherapy - resistant as characteristic of cancer stem cells ( s . oesterreich et al . cancer res ., 53 : 4443 - 4448 ( 1993 )); and aldh recently emerging as a cancer stem cell marker ( c . jauffret et al . clin cancer res ., 16 : 45 - 55 ( 2010 )). this result suggests that the secretory proteins developed in the present invention have possibility to be cancer stem cell - related secretory proteins . pancreatic cancer is one of the most deadly human tumors with 1 - 4 % of 5 year survival rate and 5 months of median survival time , and it has an extremely poor prognosis . since 80 - 90 % of patients are diagnosed at an advanced stage of being not possible to cure by curative resection , it leads to a poor clinical outcome . in addition , anticancer therapies mainly depend on chemotherapies . therefore , development for the early diagnosis method in pancreatic cancer is urgently needed than any other human cancers . to date , therapeutic effects of several anticancer agents including 5 - fluorouracil , gemcitabine and tarceva known to be effective in pancreatic cancer are very disappointing , and there is only approximately 15 % of the response rate to chemotherapy . these facts suggest that developments for the early diagnosis method and therapeutic method to pancreatic cancer in more effective manner are urgently needed . accordingly , it would be understood that improved diagnosis methods using cancer stem cells may be proposed by development of early diagnosis of cancer and combinations with the conventional markers , given that successive studies on cancer stem cell - related secretory proteins developed in the present invention have been done . further , the present invention provides novel therapeutic targets to cancers . taken together , the present invention may provide underlying principles for cancer diagnosis and therapeutics using cancer stem cells . those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present disclosure . those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the disclosure as set forth in the appended claims . 1 . b b zhou , et al . tumour - initiating cells : challenges and opportunities for anticancer drug discovery . nat rev drug discov . 8 : 806 - 823 ( 2009 ). 2 . a . dubrovska , et al ., the role of pten / akt / pi3k signaling in the maintenance and viability of prostate cancer stem - like cell populations . proc natl acad sci usa . 106 : 268 - 273 ( 2009 ). 3 . m m ho , et al ., side population in human lung cancer cell lines and tumors is enriched with stem - like cancer cells . cancer res . 67 : 4827 - 4833 ( 2007 ). 4 . t reya , et al ., stem cells , cancer , and cancer stem cells . nature . 414 : 105 - 111 ( 2001 ). 5 . j zhou , et al ., activation of the pten / mtor / stat3 pathway in breast cancer stem - like cells is required for viability and maintenance . proc natl acad sci usa . 104 : 16158 - 16163 ( 2007 ). 6 . s v shmelkov , et al ., cd133 expression is not restricted to stem cells , and both cd133 + and cd133 − metastatic pancreatic cancer cells initiate tumors . j clin invest . 118 : 2111 - 2120 ( 2008 ). 7 . l . whitesell and s . l . lindquist ., hsp90 and the chaperoning of cancer . nat rev cancer , 5 : 761 - 772 ( 2005 ). 8 . j m . lee , et al ., the epithelial -. mesenchymal transition : new insights in signaling , development , and disease . j cell biol . 172 : 973 - 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