By Vc9Vd2 T cells, indicating that chemotherapy and Vc9Vd2 T cells have additive activity even when used at suboptimal doses.Results Resistance of Colon CICs to ChemotherapyWe have previously reported that colon cancer comprises a vast majority of Title Loaded From File differentiated cells and a small population of CICs that are responsible for tumor initiation and maintenance [28]. For this study purposes, we purified and propagated colon cancer spheres from surgical fragments of 5 patients with colon carcinoma. These cancer sphere lines were identified through the expression of CD133 and the epithelial specific antigen ESA, displayed adherence to the culture dishes in the presence of serum and subsequently differentiated into large, polygonal colon cells expressing colon epithelial markers, such as villin, suggesting that colon cancer spheres maintained the ability to in vitro differentiate in enterocyte-like cells. Most importantly, when injected subcutaneously into NOD/SCID mice, a low number of colon cancer spheres, but not sphere-derived differentiated cells, retained the capacity to form a tumor that closely resembled the human original tumor (Supporting Figure S1). CICs are characterized by high resistance to drugs and general toxins which target rapidly proliferating cells and spare the slow dividing cells, due to an up-regulation of several ATP-binding cassette transporters, active DNA-repair capacity, over-expression of anti-apoptotic molecules that cause changes in the signalling pathways controlling proliferation, differentiation and apoptosis [5]. Accordingly, exposure of 5 different colon CIC lines (CIC#1 to CIC#5) to 5-FU (2.5 and 25 mg/ml) (Figure 1A) or DXR (0.025 and 0.25 mM) (Figure 1B) for 24?2 hrs had virtually no significant cytotoxic effect, as determined by PI staining. Highest doses of 5-FU (250 mg/ml) and DXR (2.5 mM) caused low, yet detectable cytotoxicity of CIC lines ranging from 1565 to 2366 (mean 6 SD). Conversely, 5-FU and DXR were fully capable of killing 3 differentiated colon cancer cell lines DLD-1, SW620 and SW403, and 2 differentiated cell lines (CDC#3 and CDC#4) obtained from two patients (P#3 and P#4) where form the CICs lines were also obtained, with a dose-dependent increase in cytotoxicity up to 85 . The viability of untreated cells was all over 90 (Figures 1A and B).Chemotherapy Upregulates DR5 (TRAIL-R2) Death Receptor Expression on CICsTo decipher the molecular mechanisms behind chemotherapymediated sensitization of CICs to Vc9Vd2 T cells cytotoxicity, we focused on expression of mRNA encoding for molecules known to be ligands for key activating receptors on Vc9Vd2 T cells and death receptors, before and after exposure of CICs to chemotherapy agents. As shown in Figure 3, all of these molecules were constitutively expressed in CICs, although expression consistently varied amongst different CIC lines; however, no major differences were observed in all tested CIC lines for HLA-class I, ICAM-1, CD155, CD112, MICA/B and ULPBP1? expression before and after exposure to chemotherapy agents. Expression of Fas (CD95), TNF-R1, DR4 (TRAIL-R1) and DR5 (TRAIL-R2) death receptors was increased in the 23977191 majority of CIC lines following exposure to chemotherapeutic agents (Figure 3), but increased expression of Fas, TNF-R1 and DR4 did not attain statistical significance. The greatest and significant increase was only observed for DR5 expression after exposure of CICs to 5-FU and, although at a lesser Title Loaded From File extent, DXR (Figure 3). Up.By Vc9Vd2 T cells, indicating that chemotherapy and Vc9Vd2 T cells have additive activity even when used at suboptimal doses.Results Resistance of Colon CICs to ChemotherapyWe have previously reported that colon cancer comprises a vast majority of differentiated cells and a small population of CICs that are responsible for tumor initiation and maintenance [28]. For this study purposes, we purified and propagated colon cancer spheres from surgical fragments of 5 patients with colon carcinoma. These cancer sphere lines were identified through the expression of CD133 and the epithelial specific antigen ESA, displayed adherence to the culture dishes in the presence of serum and subsequently differentiated into large, polygonal colon cells expressing colon epithelial markers, such as villin, suggesting that colon cancer spheres maintained the ability to in vitro differentiate in enterocyte-like cells. Most importantly, when injected subcutaneously into NOD/SCID mice, a low number of colon cancer spheres, but not sphere-derived differentiated cells, retained the capacity to form a tumor that closely resembled the human original tumor (Supporting Figure S1). CICs are characterized by high resistance to drugs and general toxins which target rapidly proliferating cells and spare the slow dividing cells, due to an up-regulation of several ATP-binding cassette transporters, active DNA-repair capacity, over-expression of anti-apoptotic molecules that cause changes in the signalling pathways controlling proliferation, differentiation and apoptosis [5]. Accordingly, exposure of 5 different colon CIC lines (CIC#1 to CIC#5) to 5-FU (2.5 and 25 mg/ml) (Figure 1A) or DXR (0.025 and 0.25 mM) (Figure 1B) for 24?2 hrs had virtually no significant cytotoxic effect, as determined by PI staining. Highest doses of 5-FU (250 mg/ml) and DXR (2.5 mM) caused low, yet detectable cytotoxicity of CIC lines ranging from 1565 to 2366 (mean 6 SD). Conversely, 5-FU and DXR were fully capable of killing 3 differentiated colon cancer cell lines DLD-1, SW620 and SW403, and 2 differentiated cell lines (CDC#3 and CDC#4) obtained from two patients (P#3 and P#4) where form the CICs lines were also obtained, with a dose-dependent increase in cytotoxicity up to 85 . The viability of untreated cells was all over 90 (Figures 1A and B).Chemotherapy Upregulates DR5 (TRAIL-R2) Death Receptor Expression on CICsTo decipher the molecular mechanisms behind chemotherapymediated sensitization of CICs to Vc9Vd2 T cells cytotoxicity, we focused on expression of mRNA encoding for molecules known to be ligands for key activating receptors on Vc9Vd2 T cells and death receptors, before and after exposure of CICs to chemotherapy agents. As shown in Figure 3, all of these molecules were constitutively expressed in CICs, although expression consistently varied amongst different CIC lines; however, no major differences were observed in all tested CIC lines for HLA-class I, ICAM-1, CD155, CD112, MICA/B and ULPBP1? expression before and after exposure to chemotherapy agents. Expression of Fas (CD95), TNF-R1, DR4 (TRAIL-R1) and DR5 (TRAIL-R2) death receptors was increased in the 23977191 majority of CIC lines following exposure to chemotherapeutic agents (Figure 3), but increased expression of Fas, TNF-R1 and DR4 did not attain statistical significance. The greatest and significant increase was only observed for DR5 expression after exposure of CICs to 5-FU and, although at a lesser extent, DXR (Figure 3). Up.

By Vc9Vd2 T cells, indicating that chemotherapy and Vc9Vd

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