Al carcinogenesis, and expecially on the 1516647 very early stages of colorectal cancer progression, identified by dysplastic aberrant crypt foci, also referred to as microadenomas [30,36]. In this context we tried to define a possible regulator of the transformations making the immune system unable to control the development of colorectal cancer at the very early stages of onset. We analyzed helper T lymphocytes, cytotoxic T lymphocytes, and natural killer T cells, identified respectively by CD4, CD8 and CD56 markers in human normal colorectal mucosa, microadenomas and carcinomas, using immunofluorescence techniques and protein quantification analyses by Western blot. In microadenomas no significant change in CD4+ cells was observed with respect to normal mucosa. On the other hand, a significant decrease of these cells in carcinomas was observed. Moreover, we noted a gradual increase of CD8+ T cells, during tumour progression. Finally a strong decrease of CD56+ cells in microadenomas was apparent, and this decrease was even more pronounced in carcinomas, where CD56+ cells were almost undetectable. We then analyzed ThPOK, a protein with a MedChemExpress Iloprost prominent role in the commitment 
of some leucocytic lineages, such as helper, cytotoxic and natural killer T cells, which have a pivotal role in defining the aggressiveness and ML 264 manufacturer prognosis of various types of cancer, including colorectal carcinomas [4,5]. ThPOK was observed to have an unexpected increase in preneoplasticThPOK and CD8+ Effector FunctionsWe subsequently analyzed the presence of effector markers, as GZMB or RUNX3, in CD8+ cells regarding to the ThPOK presence, by performing triple immunofluorescence staining. The coexpression of ThPOK and GZMB in CD8+ cells wass almost undetectable; ThPOK did not colocalize with GZMB, neither in NM, MA or CRC. The amount of GZMB decreased from NM (IFIS 59.669.1) to CRC (IFIS 26.663.7), in contrast to the increase of ThPOK since microadenomas (Figure 5, panel B). Also the levels of RUNX3 fluorescence decreased from NM (IFIS 59.669.6) to MA (IFIS 45.366.9) and to CRC (IFIS 20.8612.2) (Figure 5, panel C). In all the samples the levels of RUNX3-ThPOK-coexpressing CD8+ T cells were lower with respect to the levels of RUNX3 positive CD8+ T cells. This was more evident in MA, where there was a maximum level of RUNX3-positive CD8+ T cells. ThisThPOK in Colorectal CarcinogenesisFigure 3. Confocal immunofluorescence staining. Examples of confocal analysis of cryosections of normal colorectal 15755315 mucosa (NM), microadenoma (MA), and colorectal carcinoma (CRC), labelled by DAPI (blue), ThPOK (red), CD4 (green), CD8 (green), and CD56 (green). Double immunolabelled cells appear as yellow spots. Panels A-C: Colocalization imaging of ThPOK with CD4 in NM (panel A), MA (panel B) and CRC (panel C). Panels D-F: Double immunolabelling performed by ThPOK and CD8 in NM (panel D), MA (panel E) and CRC (panel F). Panels G-I: Immunostaining with ThPOK and CD56 in NM (panel G), MA (panel H) and CRC (panel I). Scale bar = 80 mm. doi:10.1371/journal.pone.0054488.gTable 1. Immunofluorescence quantification by confocal analysis.CD4 IFIS (mean 6 SEM) NM MA CRC 26.6163.26 27.2162.31 13.3562.59*CD8 IFIS (mean 6 SEM) 17.2262.64 30.7463.56* 46.2566.42*CD56 IFIS (mean 6 SEM) 63.94611.98 24.3265.18* 8.0663.31*ThPOK IFIS (mean 6 SEM) 24.963.0 44.6965.64* 45.4165.02*Fluorescence quantification (ImmunoFluorescence Intensity Score, IFIS, see Materials and Methods) of CD4, CD8, CD56 and ThPOK in normal colorect.Al carcinogenesis, and expecially on the 1516647 very early stages of colorectal cancer progression, identified by dysplastic aberrant crypt foci, also referred to as microadenomas [30,36]. In this context we tried to define a possible regulator of the transformations making the immune system unable to control the development of colorectal cancer at the very early stages of onset. We analyzed helper T lymphocytes, cytotoxic T lymphocytes, and natural killer T cells, identified respectively by CD4, CD8 and CD56 markers in human normal colorectal mucosa, microadenomas and carcinomas, using immunofluorescence techniques and protein quantification analyses by Western blot. In microadenomas no significant change in CD4+ cells was observed with respect to normal mucosa. On the other hand, a significant decrease of these cells in carcinomas was observed. Moreover, we noted a gradual increase of CD8+ T cells, during tumour progression. Finally a strong decrease of CD56+ cells in microadenomas was apparent, and this decrease was even more pronounced in carcinomas, where CD56+ cells were almost undetectable. We then analyzed ThPOK, a protein with a prominent role in the commitment of some leucocytic lineages, such as helper, cytotoxic and natural killer T cells, which have a pivotal role in defining the aggressiveness and prognosis of various types of cancer, including colorectal carcinomas [4,5]. ThPOK was observed to have an unexpected increase in preneoplasticThPOK and CD8+ Effector FunctionsWe subsequently analyzed the presence of effector markers, as GZMB or RUNX3, in CD8+ cells regarding to the ThPOK presence, by performing triple immunofluorescence staining. The coexpression of ThPOK and GZMB in CD8+ cells wass almost undetectable; ThPOK did not colocalize with GZMB, neither in NM, MA or CRC. The amount of GZMB decreased from NM (IFIS 59.669.1) to CRC (IFIS 26.663.7), in contrast to the increase of ThPOK since microadenomas (Figure 5, panel B). Also the levels of RUNX3 fluorescence decreased from NM (IFIS 59.669.6) to MA (IFIS 45.366.9) and to CRC (IFIS 20.8612.2) (Figure 5, panel C). In all the samples the levels of RUNX3-ThPOK-coexpressing CD8+ T cells were lower with respect to the levels of RUNX3 positive CD8+ T cells. This was more evident in MA, where there was a maximum level of RUNX3-positive CD8+ T cells. ThisThPOK in Colorectal CarcinogenesisFigure 3. Confocal immunofluorescence staining. Examples of confocal analysis of cryosections of normal colorectal 15755315 mucosa (NM), microadenoma (MA), and colorectal carcinoma (CRC), labelled by DAPI (blue), ThPOK (red), CD4 (green), CD8 (green), and CD56 (green). Double immunolabelled cells appear as yellow spots. Panels A-C: Colocalization imaging of ThPOK with CD4 in NM (panel A), MA (panel B) and CRC (panel C). Panels D-F: Double immunolabelling performed by ThPOK and CD8 in NM (panel D), MA (panel E) and CRC (panel F). Panels G-I: Immunostaining with ThPOK and CD56 in NM (panel G), MA (panel H) and CRC (panel I). Scale bar = 80 mm. doi:10.1371/journal.pone.0054488.gTable 1. 
Immunofluorescence quantification by confocal analysis.CD4 IFIS (mean 6 SEM) NM MA CRC 26.6163.26 27.2162.31 13.3562.59*CD8 IFIS (mean 6 SEM) 17.2262.64 30.7463.56* 46.2566.42*CD56 IFIS (mean 6 SEM) 63.94611.98 24.3265.18* 8.0663.31*ThPOK IFIS (mean 6 SEM) 24.963.0 44.6965.64* 45.4165.02*Fluorescence quantification (ImmunoFluorescence Intensity Score, IFIS, see Materials and Methods) of CD4, CD8, CD56 and ThPOK in normal colorect.
