Patent Application: US-67900208-A

Abstract:
chemokine receptor ccr4 and its ligands ccl1 7 and ccl22 are used as markers for the identification and / or staging of cancer . the level of ccr4 , ccl17 and ccl22 are found to increase during malignant tumour progression . ccr4 , ccl17 and ccl22 are used as markers for the stratification of cancer patients according to their suitability for treatment with anti - cancer agents . information of diagnostic character is provided by measuring the level of one or more of ccr4 , ccl 17 and ccl22 present in a patient sample . methods of treatment of cancer patients which agents that modulate the activity of ccr4 , ccl17 and ccl22 . methods of screening for agents which modulate the biological activities of ccr4 , ccl 17 and ccl22 provide anti - cancer agents .

Description:
the inventors have discovered that chemokine receptor ccr4 expression is an early event in carcinogenesis in certain tumour types . epithelial expression of a receptor for homeostatic chemokines usually present in a tissue may confer a survival advantage on the initiated cell . the chemokine receptor ccr4 was present on dysplastic non - invasive lesions of the cervix and oesophagus . this was particularly striking in some of the oesophageal cancer samples where ccr4 positive dysplastic areas were clearly seen adjacent to normal epithelial areas in the same section ( e . g . fig7 c and d ). the chemokine receptor ccr4 increased with malignant progression of the cervix . this was not only due to increased infiltration of ccr4 - positive macrophages and treg cells , but also to acquisition of ccr4 expression by epithelial cells . an unexpected finding was that ccr4 was strongly expressed on non - invasive epithelial cells in intraepithelial ( cin ) lesions as well as invasive cancer cells . progression from cin to invasive disease was associated with increased stromal cell expression of ccr4 ligands ccl17 and 22 and these chemokines stimulated growth and migration of a ccr4 - positive cervical cancer cell line ( e . g . fig4 and fig6 ). ccr4 was also detected on dysplastic as well as invasive epithelial cells in oesophageal cancer , again with ccl17 and ccl22 levels increasing during malignant progression . changes in ccl17 and 22 gradients aid transition from pre - invasive to invasive disease and attract tumour - promoting leucocytes that help initiated cells evade immune surveillance . the two ccr4 - binding chemokines , ccl17 and ccl22 , were also found on the surface of blood and lymphatic vessels in the tumour biopsies . it was not possible to quantify this but preliminary observations indicate an increase in the intensity of staining with malignant progression . another element of the ccr4 system is the non - signalling chemokine receptor d6 that has a high affinity for ccl17 and ccl22 [ 18 ]; its presence in the tissues would be expected to influence gradients of these chemokines [ 19 ]. fig6 shows that the ccr4 receptor is functional on the cervical cancer cell line c - 41 . ccr4 can be up - regulated by the microenvironment . ccr4 positive and negative cells were exposed to a number of cytokines ( tnf - α , ifn - γ , il - 4 and il - 10 ) known to be present in the cervical microenvironment and for which receptors were likely to be present on the tumour cells . none of these influenced ccr4 expression . however , ccr4 mrna levels , but not protein levels , were up - regulated by co - culture of c - 41 cells with macrophages . ccr4 and d6 are located on chromosome 3p close to where critical cervical cancer tumour suppressor genes are thought to be located with complex aberrations ( loss of heterozygosity , homozygosity and gene amplification ) reported [ 25 , 26 , 27 ]. while neither ccr4 nor d6 are directly implicated in these changes [ 26 ], genetic alterations nearby may have an impact on their regulation . ebv - immortalised b cells secrete ccl22 as well as ccl3 and ccl4 [ 28 ]. stable expression of the ebv oncogene lmp1 also induced ccl17 and ccl22 in a b cell line and lmp1 - induced ccl17 and ccl22 expression was regulated by nf - kb . it was suggested that induction of these two chemokines by ebv helps malignant cells evade immune surveillance by attracting th2 and treg cells . other oncogenic changes may induce ccl17 and ccl22 production by epithelial cells . the inventors undertook the detailed quantitation of two components of the mononuclear infiltrate in cervical cancer , specifically cd68 + macrophages and foxp3 + tregs . the density of ccr4 - positive infiltrating cells increases in cin compared with normal cervix and increases further in both scc and adenocarcinomas . cd68 + macrophages follow the same pattern and we found that these were ccr4 positive . cross talk between macrophages and malignant cells is critical at all stages of cancer progression , influencing malignant cell survival , aiding the angiogenic switch , polarizing leucocytes and aiding malignant cell invasion [ 29 , 30 , 31 ]. in cancers of the cervix and oesophagus , the chemokines ccl17 and ccl22 play a role in macrophage recruitment whereas in other cancers e . g . ovarian cancer , chemokines such as ccl2 are critical [ 32 ]. ccl17 and ccl22 are also important in the recruitment of tregs that increase in a manner parallel to the cd68 + cells in the cervical biopsies . the recruitment of treg cells to the pre - malignant and malignant lesions fosters immune privilege . for instance , in hodgkin &# 39 ; s lymphoma , hl , the malignant cells are surrounded by a large number of ccr4 + foxp3 + lymphocytes [ 33 ]. these cells , recruited by the malignant hl cells , create a favourable environment for malignant cells to escape the host immune system . the inventors think that this is also the case for cervical and oesophageal cancer . hence not only do the changes in ccl17 and cll22 gradients directly encourage tumor cell survival and spread but they attract in leucocytes that may also provide survival factors for the tumor cells and contribute to immune privilege / immunosuppression that prevents effective host responses against the tumor . these data demonstrate that the presence of epithelial ccr4 is both a highly sensitive and highly specific biomarker for both pre - malignant and malignant cervical neoplasia . the role of ccr4 expression in cervical cancer progression is , as yet , unclear though the inventors &# 39 ; data suggests that ccr4 may offer cells protection from apoptotic stimuli within the tumour environment as well as being necessary for tumour cell invasion of the basement membrane . due to its high sensitivity and selectivity , there is the potential for ccr4 to be used as a diagnostic biomarker for all stages of cervical cancer . subsequently the inventors also tested for the expression of ccr4 in 31 samples of oesophageal tumours , another tumour type that has a strong link with inflammation . by ihc they found that ccr4 was not detectable in any normal epithelial oesophageal tissue , but was present in epithelial cells of all pre - invasive and invasive lesions . due to its high sensitivity and selectivity , there is the potential for ccr4 to be used as a diagnostic biomarker for all stages of oesophageal cancer . in summary , the chemokine receptor ccr4 and its ligands increase during malignant progression of cervical , oesophageal , kidney , brain , ovarian or breast cancers . changes in ccr4 and gradients of its ligand have several pro - tumor implications . first ccr4 stimulation increases the growth and survival of the initiated and invasive cancer cells ; second , changes in chemokine gradients assists in invasion of the basement membrane and subsequent movement of the malignant cells into the blood vessels or lymphatic system . finally ccl17 and cll22 attract the types of cells , including m2 macrophages and foxp3 tregs that encourage tumor growth and allow the initiated cells to escape immune surveillance . ccr4 and its ligands may be useful diagnostic markers and therapeutic targets in epithelial neoplasia . the invention is in part described by way of experimental work and examples , in which the following materials and methods were employed : for the mrna studies , fifteen tumour biopsies from patients with cervical cancer ( 11 squamous cell carcinoma , s1 - s11 , and 4 adenocarcinomas , a1 - a4 ) and 14 samples of non - neoplastic cervical tissue ( n1 - n14 ) were obtained during surgery and snap - frozen in liquid nitrogen . diagnosis was made by the pathology department of barts and the london nhs trust . patient samples were divided according to the figo classification ( stage i , ii , iii , iv ) and tumour biopsies were classified according to increasing grade of nuclear atypia ( 1 , 2 , 3 ) or as well , moderately , or poor differentiation . for immunohistochemistry , paraffin embedded samples ( n = 166 ) from 150 different patients were obtained from barts and the london nhs trust and the clinical centre of serbia , belgrade . access to fresh and paraffin - embedded human samples satisfied the requirements of the east london and city health authority research ethics subcommittee ( lrec no . t / 02 / 046 ). resected specimens from thirty - one patients with primary squamous oesophageal carcinoma were also included in this paper . these patients were from a high - risk area for oesophageal carcinoma in anyang city , henan province , china . all patients received surgical treatment at the department of surgery of the central hospital of anyang . none of these patients had undergone chemotherapy , radiotherapy or immunomodulatory therapy before surgery . samples were taken from macroscopically cancerous and the corresponding normal areas of the same cancer patient . the tissues were fixed in pbs containing 10 % neutral - buffered formalin . cervical tissue biopsies were homogenised using a liquid nitrogen - cooled mill 6750 ( glen creston ltd , stanmore ) and then solubilised in tri reagent ™ ( sigma , poole , uk ). extracted rna was treated with 10 units dnase ( pharmacia , st albans , uk ) following the manufacturers instructions . rpa was performed using riboquant ® hcr5 and hcr6 template sets ( bd pharmingen , oxford , uk ) and [ α 32 p ] utp ( amersham international plc , aylesbury , uk ). rnase - protected fragments were run on an acrylamide - urea sequencing gel ( biorad laboratories ltd , hemel hempstead , uk ), adsorbed to filter paper and dried under vacuum . autoradiography was performed using kodak biomax ms film with a transcreen le intensifying screen ( sigma ). paraffin - embedded cervical tissues were cut under rnase - free conditions and mounted onto uv - treated palm ® membrane slides ( palm , microlaser technologies , germany ). these were then deparaffinised in xylene and rehydrated through graded alcohols . samples were stained for 1 min with mayer &# 39 ; s haematoxylin solution , dehydrated and air - dried before processing . sections were laser - microdissected following the manufacturer &# 39 ; s protocol . briefly , areas of interest were laser microdissected and catapulted into a microfuge cap containing protein kinase ( pk ) buffer . approximately 500 - 5000 cells were captured in each session . laser microdissected cells were dissolved in 100 μl pk buffer mixed with 5 μl pk . total rna was then extracted using the paraffin block rna isolation kit ( 1902 , ambion , usa ) according to the manufacturer &# 39 ; s instructions . cdna was amplified as described above and analysed using custom - made microfluidic gene array cards ( pe applied biosystems ) according to the manufacturer &# 39 ; s instructions . the gene expression profile of individual genes in seven cervical tumour samples was compared to five normal cervical samples . the gene expression levels in the normal epithelial or stromal cells samples was used as a baseline value of “ 1 ” and was compared with the average value of either tumour epithelial cells or tumour stromal cells respectively . the laser microdissected tumour samples comprised of one sample from stage 1a2 and 2b , and five of stage 1b1 . paraffin - embedded sections ( 4 μm ) were stained for ccr4 , ccl17 and ccl22 . briefly , sections were dewaxed in xylene and dehydrated through an ethanol gradient . following pbs washing the antigen was exposed using target retrieval solution ( s1700 , dako ) at 95 ° c . for 20 min or antigen unmasking solution ( h - 3300 , vector ) for 9 min in a microwave . sections were blocked with normal rabbit or goat serum for 30 min and incubated overnight at 4 ° c . with the primary antibody : ccr4 ( 1 : 300 , ab1669 , abcam , cambridge ), ccl17 ( 1 : 50 , ab9816 - 50 , abcam , cambridge ) and ccl22 ( 1 : 20 , 500 - p107 , peprotech ). following incubation with a biotinylated secondary antibody ( anti - goat or anti - rabbit igg , 1 : 200 , vector ) for 30 min at room temperature , antigens were revealed with 3 , 3 ′- diaminobenzidine ( dab ; sigma ). slides were then counterstained with haematoxylin , dehydrated and mounted . omission of the primary antibody was used as a negative control . to check specificity of the ccr4 antibody , some ccr4 - negative cells were transfected with cdna for this chemokine receptor . the ccr4 antibody detected surface protein only on the successfully transfected cells . for assessment of ccr4 , ccl17 and ccl22 expression on non - malignant and malignant epithelial cells , each sample was assessed semi - quantitatively with the following scoring system : 0 ( no positive protein expression ), + 1 (& lt ; 25 % of the cross - section on average has positive expression ), + 2 ( 26 - 50 %), + 3 ( 51 - 75 %), + 4 (& gt ; 76 %). the intensity of positive cells was analysed as follows : 0 ( no expression ), 1 ( mild expression ), 2 ( moderate expression ), 3 ( strong expression ). scoring of ccr4 , ccl17 and ccl22 expression in tumour stroma ( intratumoral infiltrating cells ) and the invasive border of the tumour ( peritumoral infiltrating cells ) was performed based on the ‘ running mean ’ method [ 43 ]. necrotic areas were avoided . a total of 15 high - power fields (× 400 magnification ) were counted . five scales were set up as follows : 0 = no ccr4 protein expression ; + 1 = 1 - 10 ccr4 positive cells per hpf ; + 2 = 10 - 20 positive cells per hpf ; + 3 ( 21 - 30 cells per hpf ); + 4 (& gt ; 30 cells ). the overall staining result was obtained by calculating ‘ percentage ’×‘ intensity ’. the ihc scoring on tumour and infiltrating cells was performed by a board - certified pathologist ( yw ). the cervical cancer cell line c - 41 ( atcc , rockville , md ., usa ) was cultured in dmem medium supplemented with 10 % fcs . in some experiments cells were stimulated with 1 , 10 , 100 , or 1000 ng / ml of ccl17 or ccl22 ( peprotech , london , uk ). proliferation and migration were assessed using methods described previously [ 11 ]. statistical significance was evaluated using unpaired t - test with welch &# 39 ; s correction ( instat software , san diego , calif .). a p value of & lt ; 0 . 05 was considered significant . ribonuclease protection assays ( rpa ) were used to screen for 13 chemokine receptor mrnas in fresh - frozen biopsies of human cervical tissue . as can be seen from the summary graph in fig1 a , a range of chemokine receptor mrnas was found in cervical tissue extracts with some discrete differences between the non - neoplastic and the malignant biopsies . of particular interest was the chemokine receptor ccr4 , which was present in the malignant cervix but not in extracts from non - neoplastic cervical biopsies ( fig1 b ). as chemokine receptor expression was examined on whole tissue extracts containing a mixed population of stromal cells and epithelial cells , we next investigated the cellular source of the ccr4 mrna . mrna was extracted from laser microdissected stromal and epithelial cell areas of normal and malignant cervical biopsies , and semi - quantitative real time rt - pcr was used to analyse ccr4 expression with 18s rrna as a control . as shown in fig1 c , ccr4 mrna was up - regulated in stromal areas from malignant tissues when these were compared to their non - neoplastic counterparts . in addition , and unexpectedly , ccr4 mrna was also up - regulated in extracts from the malignant epithelial cell areas compared to normal epithelium . to investigate further these observations relating to ccr4 mrna , we stained a cohort of biopsies for ccr4 using immunohistochemistry , ihc . we assessed ccr4 protein expression in 166 samples of paraffin embedded cervical tissues from 150 different patients : nonneoplastic , n = 23 ; cin i , n = 30 ; cin ii , n = 17 ; cin iii , n = 16 ; scc , n = 45 ; recurrent tumour , n = 15 ; lymph node metastasis ( ln mets ), n = 10 ; adenocarcinoma , n = 10 . both leucocytes and epithelial cells expressed ccr4 protein . to quantify our results , an ihc score was calculated by multiplying the ‘ positivity ’ and ‘ intensity ’ ( see the description of methods and fig8 and 9 for a more details ). experiment 2 — ccr4 protein is found on infiltrating leucocytes in human cervical biopsies . as shown in fig2 ( a - c ), leucocytes in the stromal areas of the biopsies stained positive for ccr4 . the ihc score for ccr4 positivity in the stromal areas is summarised in fig3 a ( white bars ). the non - neoplastic tissues were negative for ccr4 leucocytes ( fig2 a , 3 a ). there were more ccr4 expressing stromal cells in the cin samples ( fig2 b , 3 a ) and this increased further in the invasive neoplastic cervical samples ( fig2 c , 3 a ). the intensity of ccr4 expression on the infiltrating leucocytes also increased with malignant progression . in non - neoplastic tissues this was mild ; intensity was moderate to strong in cin and adenocarcinomas , and intensity was strong in invasive scc , recurrent tumours and lymph node metastases ( fig8 ). the stroma consists of various cell types , and tests were carried out to ascertain which of the infiltrating cells contributed to ccr4 expression . as macrophages and treg cells express ccr4 , ccr4 protein expression was examined in these two cell types and also counted the number of cd68 + macrophages and foxp3 + treg cells in the tissue biopsies were counted . the number of cd68 + macrophages and foxp3 + tregs increased with malignant progression of the cervix . as shown in fig2 d - i there were few cd68 and foxp3 cells in biopsies of normal cervix , but the numbers increased in cin and both cell types were prominent in invasive cancers . the numbers of cd68 + and foxp3 + cells were then counted in the 122 and 33 biopsies respectively . as shown in fig3 b there was a significant ( p & lt ; 0 . 001 ) increase in cd68 + cells in cin lesions compared to normal cervix . the number of 13 cd68 + cells further increased in scc , adenocarcinoma , recurrent cancers and lymph node metastases ( p & lt ; 0 . 001 , and for ln mets p & lt ; 0 . 05 , compared to normal cervix ). a similar increase in foxp3 + cells occurred with malignant progression with scc , adenocarcinomas and lymph node metastases all showing significant increases in the foxp3 + infiltrate compared to normal cervix ( p & lt ; 0 . 01 ). to study the phenotype of infiltrating ccr4 expressing cells , an assessment was made of cell surface expression of ccr4 by macrophages and tregs using double immunohistochemical staining for cd68 and foxp3 . this confirmed that cd68 + and foxp3 + cells also express ccr4 protein ( data not shown ). a subset of 33 paraffin embedded tissues were also stained for scavenger receptor - a ( sr - a ) protein ( non - neoplastic = 10 , cin = 10 , scc = 10 , adenocarcinoma = 3 ). sr - a is a cell surface marker for m2 alternatively activated macrophages [ 12 ]. sr - a could not be detected on stromal cells in normeoplastic lesions but in cin and invasive cancer , a proportion of the cd - 68 + cells expressed sr - a ( data not shown ). these studies show , firstly , that malignant progression of the cervix is associated with an increase in the numbers of cd68 + macrophages and foxp3 + treg cells . these cells could provide pro - tumour growth factors for the malignant cells and also help create an immunosuppressive microenvironment that would help transformed cells evade immune surveillance . secondly , these results showed that the original observation of an increase in ccr4 mrna in malignant compared to normal cervical cancer was due , at least in part , to an increased infiltrate of ccr4 expressing leucocytes , including macrophages and treg cells . however , the laser microdissection result showed that ccr4 mrna was also increased in epithelial areas of the tumours , and when assessing ccr4 protein on infiltrating leukocytes , it was clear that the chemokine receptor was also present on some epithelial cells ( see fig2 b and c ). this was unexpected and warranted further investigation . in the non - neoplastic cervical biopsies , normal epithelial cells did not express ccr4 ( fig2 a ). however epithelial cells in over 90 % of the cin cases expressed ccr4 ( fig2 b and 2e ). 96 % of the scc samples had ccr4 - positive epithelial cells ( fig2 c and 2f ) and epithelial cells 90 % of adenocarcinoma samples were positive for ccr4 ( fig2 g ). malignant epithelial cells in all recurrent tumours and lymph node metastases expressed ccr4 protein . full details of the ihc results for ccr4 protein on epithelial cells are shown in fig9 and summarised in fig2 d ( black bars ). ccr4 expression was not restricted to a minority of epithelial cells . fig2 e - g and 9 show that the majority of malignant epithelial cells in cervical biopsies of cin and invasive cancer were ccr4 positive . more detail relating to the ihc score for different stages of cin is shown in fig2 h . this shows that epithelial ccr4 expression was essentially unchanged through progression from cini to iii but that stromal levels of ccr4 increased from cin i - iii statistical analysis of the data in fig8 and 9 showed that cin lesions showed a significant up - regulation of ccr4 protein in both epithelial ( p = 0 . 0001 ) and stromal ( p = 0 . 0001 ) compartments when compared to non - neoplastic cervical tissues . ccr4 expression in invasive scc was also significantly increased in both the epithelial ( p = 0 . 0001 ) and stromal compartments ( p = 0 . 0001 ) when compared to non - neoplastic tissues . also in adenocarcinoma samples , ccr4 was up - regulated 15 on epithelial ( p = 0 . 0006 ) and stromal cells ( p = 0 . 0050 ) when compared to non - neoplastic cervical tissue . in normal tissues , ccl22 is a product of macrophages , monocytes , dc , b and t cells [ 13 , 14 ]. it is also found in epithelial tissues ; for instance intestinal epithelium constitutively produces ccl22 that can be further up - regulated by inflammatory cytokines such as tnf - [ 15 ]. mrna was isolated from 14 biopsies of normal cervix , 11 scc and 4 adenocarcinomas and ccl22 levels assessed by real time rt pcr . as shown in fig4 a ccl22 mrna levels were lower in the malignant tissues compared with the normal biopsies but this was not significant ( p = 0 . 43 ). a total of 52 samples of paraffin embedded cervical tissues from 50 different patients were assessed for ccl22 protein : non - neoplastic , n = 16 ; cin , n = 17 ; scc , n = 19 . in all cervical biopsies , ccl22 was detected in the epithelial cells ( fig4 b - d ). fourteen of 16 normal samples , 15 / 17 cin , 14 / 19 scc had ccl22 positive epithelial cells . infiltrating leucocytes in all biopsies contained ccl22 ( fig4 c , d ). the epithelial ihc score declined slightly between the cin lesions and sccs ( fig4 e black bars ). however , the stromal score for ccl22 increased from normal to cin and scc ( fig4 e white bars ). in normal tissues , ccl17 is expressed by vascular and lymphatic endothelial cells but is also produced by macrophages , dc and keratinocytes [ 16 , 17 , 55 ] mrna was isolated from 14 biopsies of normal cervix , 11 scc and 4 adenocarcinomas and ccl17 levels assessed by real time rt - pcr . as shown in fig5 a levels of ccl17mrna were higher in scc compared to normal cervix . a total of 74 samples of paraffin embedded cervical tissues from 70 different patients were assessed for ccl17 protein : non - neoplastic , n = 21 ; cin , n = 33 ; scc , n = 20 . normal cervical biopsies had low levels of ccl17 in a minority of samples both the epithelium and stroma ( fig5 b ). only 2 / 19 normal samples had ccl17 positive cells in the epithelium compared with 23 / 33 cin samples and 13 / 20 scc . the number of stromal cells that were ccl17 positive was increased in cin ( fig5 c ) and scc ( fig5 d ) compared to normal samples . six of 21 normal biopsies had ccl17 positive stromal cells compared to 25 / 33 cin and 15 / 20 scc . the epithelial and stromal ccl17 ihc score was increased in cin and scc compared to normal biopsies ( fig5 e ). when the ihc scores from individual biopsies were analysed , there was a statistically significant difference in ccl17 ihc score in stroma from cin ( p = 0 . 001 ) and scc ( p = 0 . 002 ) compared to normal biopsies . there was also a difference in the ihc scores in epithelial areas of cin ( p = 0 . 001 ) and scc ( p = 0 . 009 ) compared to normal . these data show that chemokine gradients changed with the transition from intraepithelial neoplasia to invasive disease . to investigate the biological significance of ccr4 expression on cervical cancer cells a number of cervical cancer cell lines ( caski , me180 , hela - ohio , hela - s3 , siha , c33a , c41 - 1 ) were screened for ccr4 expression . the cell line c - 41 expressed cell surface ccr4 in a constitutive manner ( fig6 a ). using facs analysis it was shown that c - 41 cells expressed cell surface ccr4 . this cell line also had intracellular ccl22 protein but the other ccr4 ligand ccl17 was not present ( fig6 a ). following stimulation with 100 ng / ml ccl22 , cell surface ccr4 protein was internalized on c - 41 cells after 2 hours and returned back to the surface after 3 hours ( fig6 b ). following stimulation with 100 ng / ml ccl17 , cell surface ccr4 protein was also internalized on c - 41 cells after 2 hours and returned back to the surface after 3 hours ( fig6 c ). c - 41 cells demonstrated a typical bell - shaped chemotactic response towards both ccl17 and ccl22 in trans - well migration assays ( fig6 d ). at 10 ng / ml , ccl17 induced significant migration ( p = 0 . 036 ) and also at 100 ng / ml and 1000 ng / ml ( p = 0 . 0006 and p = 0 . 0004 respectively ). similar results were seen with ccl22 at 10 ng / ml ( p = 0 . 0081 ), 10 ng / ml ( p = 0 . 0009 ) and at 1000 ng / ml ( p = 0 . 0348 ). c - 41 cells showed increased proliferation after stimulation with either 10 ng / ml ( p = 0 . 017 ) or 100 ng / ml ( p = 0 . 044 ) of ccl17 . 1 ng / ml ccl22 ; ( p = 0 . 026 ), 100 ng / ml ccl22 ( p = 0 . 043 ), but not 10 ng / ml ccl22 ( p = 0 . 195 ), also simulated c - 41 cell growth ( fig5 c and d ). ccr4 was therefore functional on this cervical cancer cell line , suggesting that it may also be functional in vivo . experiment 9 — ccr4 is expressed on epithelial and stromal cells during malignant progression of the oesophagus it was unknown as to whether the expression of ccr4 and changes in chemokine ligand were specific for cervical cancer or whether they were seen in any other epithelial malignancies that have a link with inflammation . cancer of the oesophagus is an epithelial cancer where examples of all stages of neoplastic progression can be readily obtained , often simultaneously from the same patient . ccr4 expression was examined in 31 specimens from patients with oesophageal cancer . in 27 of the cases , all stages of carcinogenesis of the oesophagus : normal , hyperplasia , dysplasia , in situ carcinoma and invasive cancer , were present in biopsies from the same patient . four of 31 cases had pre - invasive lesions without invasive cancer areas . as shown in fig7 a , there was no detectable ccr4 expression in normal epithelial cells of oesophagus , apart from a few ccr4 - positive cells around the basal layer of hyperplastic epithelium ( fig7 b ). in 30 of 31 cases ccr4 protein was present on epithelial cells in all stages of pre - invasive lesions ( fig7 c , d ), that the intensity and percentage of ccr4 expressing cells in dysplastic lesions was much higher than in hyperplastic epithelial cells . epithelial cells in the invasive cancer were also ccr4 - positive ( fig7 h ). in some places , there was an abrupt transition between normal and abnormal mucosa fig7 c , d ). most interestingly , there were high levels of ccr4 expression in the dysplastic cells , but the cells in the superficial layers , and adjacent normal mucosa were negative . ccr4 - positive cells were also present in the stroma , the pattern being the same as in cervical cancer . as shown in fig7 e , there were few ccr4 - positive cells in the normal submucosa ; with malignant progression , there were more ccr4 positive cells infiltrating the stroma ( fig7 f - h ). ihc was used to assess ccl17 and ccl22 expression in 23 of the oesophageal samples in which all stages of carcinogenesis were present in each sample . ccl17 was generally absent in both the epithelial and stromal areas of the normal tissues , although there were a few ccl17 - positive cells in the stroma and a minority of hyperplastic areas . the number of samples continuing ccl17 - positive epithelial or stromal cells increased in dysplasia and was highest in invasive areas with 10 / 23 of these showing some ccl17 positivity . of particular note was strong ccl17 immunoreactivity on the endothelial cells or blood / lymphatic vessels in the submucosa of dysplastic but not normal epithelium . similar to the observations in cervical cancer , the levels of stromal positivity for ccl22 also increased with malignant progression . only 1 / 23 samples showed ccl22 positive cells in the stroma of the normal areas , but in dysplastic areas and invasive areas 20 / 23 and 18 / 23 samples respectively contained ccl22 - positive cells in the stroma . the stromal cell ccl22 positivity increased with the degree of dysplasia . eight of 23 dysplasia i samples had ccl22 positive cells in the stroma ; this increased to 19 of 23 samples of dysplasia ii and 20 / 23 samples of dysplasia iii . there was one difference between the cervical and oesophageal epithelium in that ccl22 was not detected in normal epithelium although it has been reported to be present in normal intestinal epithelium [ 15 ]. epithelial ccl22 expression increased with malignant progression of the oesophagus ; 0 / 23 samples were positive in the normal areas , 2 / 23 hyperplasias , 7 / 23 dysplasias and 14 / 23 invasive areas had ccl22 positive epithelial cells . finally , more endothelial cells of blood vessels within the stroma of invasive cancer tissues were positive for ccl22 staining compared with normal and dysplastic epithelium . experiment 11 — the results of a screen for ccr4 expression in a wider range of tumours using a tumour cdna library ( cancer research uk ) containing cdna generated from rna isolated from 5 - 10 tumour samples and 2 - 5 normal samples for 11 different tumour types : lung , colon , bladder , stomach , pancreas , skin , breast , brain , oesophagus , ovary and prostate . the ccr4 mrna expression levels were measured using quantitative real time rt - pcr . ccr4 mrna levels were significantly elevated in cancers of the cervix , oesophagus , kidney , brain , breast and ovary . experiment 12 — analysis of expression of ccr4 in cervical and renal cancer cell lines fig1 shows the results of fluorescence activated cell scanning ( facs ) analysis on cervical ( c41 , c33a ) and renal cancer cell lines using an anti - ccr4 antibody to detect ccr4 expression . all the cell lines expressed ccr4 . the dashed lines in fig1 show the data for an isotype - matched control antibody . experiment 13 — effects of common cytokines on ccr4 expression by tumour cells the most common cytokines present in a tumour are il - 10 , tgf - β , fgf , tnf - α . the c41 cervical cancer cell line was stimulated in culture with different cytokines ( il - 10 , tnf - α , tgf - β , fgf ; 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