Iotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions and incubated with PE-conjugated antibody Foxp3 or IgG1 (Miltenyi Biotec, Bergisch Gladbach, Germany) for 30 min at 4uC in the dark. The fluorescence of Foxp3 was measured and analyzed with a FACS flow cytometer (Coulter EPICS XL, Beckman Coulter, Brea, USA).Immunohistochemical and immunofluorescent stainingCD4 and CD25 antibodies were provided by Dako (Hamburg, Germany). Two different Foxp3 antibody clones (ab22510 mouse monoclonal and ab2481 goat polyclonal) were purchased from Abcam (Cambridge, UK), TGF-b antibody was provided by Serotec (Duesseldorf, Germany), and IL-10 antibody by R D Systems (Wiesbaden-Nordenstadt, Germany). Isotype controlantibodies were purchased from eBioscience (San Diego, USA). Secondary antibodies used for immunofluorescence double staining and immunohistochemistry were provided by Jackson ImmunoResearch Laboratories Inc. (Suffolk, England). Secondary EPCAM and CD4 antibodies were FITC-conjugated AffiniPure Donkey anti-goat IgG and secondary antibody of Foxp3, IL-10, and TGF-b was Cy3-conjugated AffiniPure Donkey anti-mouse IgG at a 1:200 dilution (Jackson ImmunoResearch). For cytospin preparations colorectal carcinoma cells from the patients with CRC were cultured in short-term primary cultures and established human colon cancer cell lines were harvested at an exponential growth phase using enzyme free cell dissociation solution (Merck Millipore, Met-Enkephalin Billerica, MA). After washing with dPBS (Life Technologies, GIBCO, Carlsbad, CA) twice, cells were adjusted to a concentration of 26105 cells/ml. Cytospins were performed with 50 ml cell suspension at 550 rpm for 1 min in a Cytospin4 Cytocentrifuge (Thermo Fisher Scientific, Waltham, MA). The staining of CD4, CD25, Foxp3, TGF-b, and IL-10 was performed on serial cryostat sections of the 65 snap-frozen CRC specimens in early-stage tumors (UICC I/II) and late-stage tumors (UICC III/IV) with neighbouring normal colon tissue and 10 normal colon specimens. All tumors stained positive for cytokeratin-20 (CK-20) (Dako, Hamburg, Germany) and negative for cytokeratin-7 (CK-7) (Dako), a pattern characteristic for colonic adenocarcinoma [32]. First we assessed H.E. sections from each tumor tissue to differentiate between cancer cell areas, stromalFoxp3 Expression and CRC Disease ProgressionTable 3. Clinicopathological characteristics of the study population and discrimination of Foxp3 expression profiles.TotalFoxp3 (cancer cells) Low High 34 (100 ) 64.265.9 n.s. TestFoxp3 (Treg) Low 38 (100 ) 64.266.0 High 27 (100 ) 63.865.6 n.s. TestCases (n) Age (y; mean 6 sd) Gender Male CASIN chemical information Female Primary tumor Colon Rectum Differentiation G1 G2 G3/4 Depth of invasion pT1 pT2 pT3 pT4 Lymph node metastasis pN0 pN1? UICC stage UICC I UICC II UICC III UICC IV Mean OS (m) Median OS (m) Log-Rank test65 (100 ) 64.065.31 (100 ) 63.965.37 (57 ) 28 (43 )18 (58 ) 13 (42 )19 (56 ) 15 (44 )n.s.21 (55 ) 17 (45 )16 (59 ) 11 (41 )n.s.26 (40 ) 39 (60 )12 (39 ) 19 (61 )14 (41 ) 20 (59 )n.s.16 (42 ) 22 (58 )10 (37 ) 17 (63 )n.s.12 (18 ) 31 (48 ) 22 (34 )7 (22 ) 16 (52 ) 8 (26 )5 (15 ) 15 (44 ) 14 (41 )n.s.4 (11 ) 21 (55 ) 13 (34 )8 (30 ) 10 (37 ) 9 (33 )n.s.14 (22 ) 23 (35 ) 17 (26 ) 11 (17 )10 (32 ) 14 (45 ) 7 (23 ) -4 (12 ) 9 (27 ) 10 (29 ) 11 (32 ),0.8 (21 ) 13 (34 ) 10 (26 ) 7 (19 )6 (22 ) 10 (37 ) 7 (26 ) 4 (15 )n.s.31 (48 ) 34 (52 )25 (81 ) 6 (19 )6 (18 ) 28 (82 ),0.14 (37 ) 24 (63 )17 (63 ) 10 (37 )0.15 (23 ) 19 (29 ) 22 (34.Iotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions and incubated with PE-conjugated antibody Foxp3 or IgG1 (Miltenyi Biotec, Bergisch Gladbach, Germany) for 30 min at 4uC in the dark. The fluorescence of Foxp3 was measured and analyzed with a FACS flow cytometer (Coulter EPICS XL, Beckman Coulter, Brea, USA).Immunohistochemical and immunofluorescent stainingCD4 and CD25 antibodies were provided by Dako (Hamburg, Germany). Two different Foxp3 antibody clones (ab22510 mouse monoclonal and ab2481 goat polyclonal) were purchased from Abcam (Cambridge, UK), TGF-b antibody was provided by Serotec (Duesseldorf, Germany), and IL-10 antibody by R D Systems (Wiesbaden-Nordenstadt, Germany). Isotype controlantibodies were purchased from eBioscience (San Diego, USA). Secondary antibodies used for immunofluorescence double staining and immunohistochemistry were provided by Jackson ImmunoResearch Laboratories Inc. (Suffolk, England). Secondary EPCAM and CD4 antibodies were FITC-conjugated AffiniPure Donkey anti-goat IgG and secondary antibody of Foxp3, IL-10, and TGF-b was Cy3-conjugated AffiniPure Donkey anti-mouse IgG at a 1:200 dilution (Jackson ImmunoResearch). For cytospin preparations colorectal carcinoma cells from the patients with CRC were cultured in short-term primary cultures and established human colon cancer cell lines were harvested at an exponential growth phase using enzyme free cell dissociation solution (Merck Millipore, Billerica, MA). After washing with dPBS (Life Technologies, GIBCO, Carlsbad, CA) twice, cells were adjusted to a concentration of 26105 cells/ml. Cytospins were performed with 50 ml cell suspension at 550 rpm for 1 min in a Cytospin4 Cytocentrifuge (Thermo Fisher Scientific, Waltham, MA). The staining of CD4, CD25, Foxp3, TGF-b, and IL-10 was performed on serial cryostat sections of the 65 snap-frozen CRC specimens in early-stage tumors (UICC I/II) and late-stage tumors (UICC III/IV) with neighbouring normal colon tissue and 10 normal colon specimens. All tumors stained positive for cytokeratin-20 (CK-20) (Dako, Hamburg, Germany) and negative for cytokeratin-7 (CK-7) (Dako), a pattern characteristic for colonic adenocarcinoma [32]. First we assessed H.E. sections from each tumor tissue to differentiate between cancer cell areas, stromalFoxp3 Expression and CRC Disease ProgressionTable 3. Clinicopathological characteristics of the study population and discrimination of Foxp3 expression profiles.TotalFoxp3 (cancer cells) Low High 34 (100 ) 64.265.9 n.s. TestFoxp3 (Treg) Low 38 (100 ) 64.266.0 High 27 (100 ) 63.865.6 n.s. TestCases (n) Age (y; mean 6 sd) Gender Male Female Primary tumor Colon Rectum Differentiation G1 G2 G3/4 Depth of invasion pT1 pT2 pT3 pT4 Lymph node metastasis pN0 pN1? UICC stage UICC I UICC II UICC III UICC IV Mean OS (m) Median OS (m) Log-Rank test65 (100 ) 64.065.31 (100 ) 63.965.37 (57 ) 28 (43 )18 (58 ) 13 (42 )19 (56 ) 15 (44 )n.s.21 (55 ) 17 (45 )16 (59 ) 11 (41 )n.s.26 (40 ) 39 (60 )12 (39 ) 19 (61 )14 (41 ) 20 (59 )n.s.16 (42 ) 22 (58 )10 (37 ) 17 (63 )n.s.12 (18 ) 31 (48 ) 22 (34 )7 (22 ) 16 (52 ) 8 (26 )5 (15 ) 15 (44 ) 14 (41 )n.s.4 (11 ) 21 (55 ) 13 (34 )8 (30 ) 10 (37 ) 9 (33 )n.s.14 (22 ) 23 (35 ) 17 (26 ) 11 (17 )10 (32 ) 14 (45 ) 7 (23 ) -4 (12 ) 9 (27 ) 10 (29 ) 11 (32 ),0.8 (21 ) 13 (34 ) 10 (26 ) 7 (19 )6 (22 ) 10 (37 ) 7 (26 ) 4 (15 )n.s.31 (48 ) 34 (52 )25 (81 ) 6 (19 )6 (18 ) 28 (82 ),0.14 (37 ) 24 (63 )17 (63 ) 10 (37 )0.15 (23 ) 19 (29 ) 22 (34.
