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Deciphering the cellular source of tumor relapse identifies CD44 as a major therapeutic target in pancreatic adenocarcinoma.

Molejon MI, Tellechea JI, Loncle C, Gayet O, Gilabert M, Duconseil P, Lopez-Millan MB, Moutardier V, Gasmi M, Garcia S, Turrini O, Ouaissi M, Poizat F, Dusetti N, Iovanna J - Oncotarget (2015)

Bottom Line: The origin and biological characteristics of residual tumor cells in PDAC still remain unclear.During PDAC relapse, proliferating CD44+ cells decrease expression of ZEB1, while overexpressing the MUC1 protein, and gain morphological and biological characteristics of differentiation.We confirmed the propagation of CD44+ cells in samples from cases of human relapse, following standard PDAC treatment.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche en Cancérologie de Marseille (CRCM), INSERM U1068, CNRS UMR 7258, Aix-Marseille Université and Institut Paoli-Calmettes, Parc Scientifique et Technologique de Luminy, Marseille, France.

ABSTRACT
It has been commonly found that in patients presenting Pancreatic Ductal Adenocarcinoma (PDAC), after a period of satisfactory response to standard treatments, the tumor becomes non-responsive and patient death quickly follows. This phenomenon is mainly due to the rapid and uncontrolled development of the residual tumor. The origin and biological characteristics of residual tumor cells in PDAC still remain unclear. In this work, using PDACs from patients, preserved as xenografts in nude mice, we demonstrated that a residual PDAC tumor originated from a small number of CD44+ cells present in the tumor. During PDAC relapse, proliferating CD44+ cells decrease expression of ZEB1, while overexpressing the MUC1 protein, and gain morphological and biological characteristics of differentiation. Also, we report that CD44+ cells, in primary and residual PDAC tumors, are part of a heterogeneous population, which includes variable numbers of CD133+ and EpCAM+ cells. We confirmed the propagation of CD44+ cells in samples from cases of human relapse, following standard PDAC treatment. Finally, using systemic administration of anti-CD44 antibodies in vivo, we demonstrated that CD44 is an efficient therapeutic target for treating tumor relapse, but not primary PDAC tumors. We conclude that CD44+ cells generate the relapsing tumor and, as such, are themselves promising therapeutic targets for treating patients with recurrent PDAC.

No MeSH data available.


Related in: MedlinePlus

Deregulation of differentiation markers in PDXs after chemotherapy(A) Immunofluorescent analysis of CD44 (green), ZEB1 (red) and DAPI (blue) in three PDX samples treated with vehicle (upper panel) or with gemcitabine (bottom panel). (B) WB analysis for ZEB1 in protein lysates from tumor samples upon vehicle or gemcitabine treatment. (C) Quantification of ZEB1 protein expression. (D) Co-localization analysis of CD44 (green), MUC1 (red) and DAPI (blue) in PDX samples treated with vehicle (upper panel) or with gemcitabine (bottom panel). (E) WB analysis for MUC1 in protein lysates from tumor samples treated with vehicle or with gemcitabine. (F) Quantification of the expression of the MUC1 protein in tumor samples. (G) Immunofluorescent labeling of PCNA (red), CD44 (green) and DAPI (blue) in PDXs vehicle or treated with gemcitabine. The CD44+/PCNA+ ratio is shown on the left side (H). Scale bar represents 10 μm. Error bars ± SEM; n=3 per group. *P<0.05, **P<0.001 compared to vehicle treatment.
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Figure 3: Deregulation of differentiation markers in PDXs after chemotherapy(A) Immunofluorescent analysis of CD44 (green), ZEB1 (red) and DAPI (blue) in three PDX samples treated with vehicle (upper panel) or with gemcitabine (bottom panel). (B) WB analysis for ZEB1 in protein lysates from tumor samples upon vehicle or gemcitabine treatment. (C) Quantification of ZEB1 protein expression. (D) Co-localization analysis of CD44 (green), MUC1 (red) and DAPI (blue) in PDX samples treated with vehicle (upper panel) or with gemcitabine (bottom panel). (E) WB analysis for MUC1 in protein lysates from tumor samples treated with vehicle or with gemcitabine. (F) Quantification of the expression of the MUC1 protein in tumor samples. (G) Immunofluorescent labeling of PCNA (red), CD44 (green) and DAPI (blue) in PDXs vehicle or treated with gemcitabine. The CD44+/PCNA+ ratio is shown on the left side (H). Scale bar represents 10 μm. Error bars ± SEM; n=3 per group. *P<0.05, **P<0.001 compared to vehicle treatment.

Mentions: To characterize the evolution of CD44-expressing cells, we investigated the expression of cell differentiation-associated markers. To this end, we used immunofluorescence to analyze the expression of the ZEB1 protein, which has been associated with poor differentiation, and CD44. We found a significant decrease of ZEB1 expression after gemcitabine treatment. Remarkably, we detected ZEB1 in 42±3.2%, 11±0.9% and 53±3.5% of cells in vehicle-treated PDXs whereas its expression decreased to 9.2±1.1%, 0.9±0.1% and 33±3.1% after gemcitabine treatment in X-IPC, C-NOR and AO-IPC PDXs, respectively (Figure 3A). ZEB1 frequently colocates with CD44 in vehicle-treated PDXs but rarely in gemcitabine-treated PDXs. The decreased expression of ZEB1 after gemcitabine treatment was confirmed by western blot analysis (Figure 3B and 3C). Furthermore, we evaluated MUC1 expression, which, conversely, is strongly associated with cellular differentiation. As illustrated in Figure 3D, though some cells of the vehicle-treated PDXs expressed MUC1, but never colocalizes with CD44+ cells. However, after gemcitabine treatment we found that the majority of the CD44+ residual cells expressed MUC1 (Figure 3D and F). Western blot analysis confirmed that MUC1 expression dramatically increased in gemcitabine-treated tumors, when compared to vehicle-treated samples (Figure 3E).


Deciphering the cellular source of tumor relapse identifies CD44 as a major therapeutic target in pancreatic adenocarcinoma.

Molejon MI, Tellechea JI, Loncle C, Gayet O, Gilabert M, Duconseil P, Lopez-Millan MB, Moutardier V, Gasmi M, Garcia S, Turrini O, Ouaissi M, Poizat F, Dusetti N, Iovanna J - Oncotarget (2015)

Deregulation of differentiation markers in PDXs after chemotherapy(A) Immunofluorescent analysis of CD44 (green), ZEB1 (red) and DAPI (blue) in three PDX samples treated with vehicle (upper panel) or with gemcitabine (bottom panel). (B) WB analysis for ZEB1 in protein lysates from tumor samples upon vehicle or gemcitabine treatment. (C) Quantification of ZEB1 protein expression. (D) Co-localization analysis of CD44 (green), MUC1 (red) and DAPI (blue) in PDX samples treated with vehicle (upper panel) or with gemcitabine (bottom panel). (E) WB analysis for MUC1 in protein lysates from tumor samples treated with vehicle or with gemcitabine. (F) Quantification of the expression of the MUC1 protein in tumor samples. (G) Immunofluorescent labeling of PCNA (red), CD44 (green) and DAPI (blue) in PDXs vehicle or treated with gemcitabine. The CD44+/PCNA+ ratio is shown on the left side (H). Scale bar represents 10 μm. Error bars ± SEM; n=3 per group. *P<0.05, **P<0.001 compared to vehicle treatment.
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Related In: Results  -  Collection

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Show All Figures
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Figure 3: Deregulation of differentiation markers in PDXs after chemotherapy(A) Immunofluorescent analysis of CD44 (green), ZEB1 (red) and DAPI (blue) in three PDX samples treated with vehicle (upper panel) or with gemcitabine (bottom panel). (B) WB analysis for ZEB1 in protein lysates from tumor samples upon vehicle or gemcitabine treatment. (C) Quantification of ZEB1 protein expression. (D) Co-localization analysis of CD44 (green), MUC1 (red) and DAPI (blue) in PDX samples treated with vehicle (upper panel) or with gemcitabine (bottom panel). (E) WB analysis for MUC1 in protein lysates from tumor samples treated with vehicle or with gemcitabine. (F) Quantification of the expression of the MUC1 protein in tumor samples. (G) Immunofluorescent labeling of PCNA (red), CD44 (green) and DAPI (blue) in PDXs vehicle or treated with gemcitabine. The CD44+/PCNA+ ratio is shown on the left side (H). Scale bar represents 10 μm. Error bars ± SEM; n=3 per group. *P<0.05, **P<0.001 compared to vehicle treatment.
Mentions: To characterize the evolution of CD44-expressing cells, we investigated the expression of cell differentiation-associated markers. To this end, we used immunofluorescence to analyze the expression of the ZEB1 protein, which has been associated with poor differentiation, and CD44. We found a significant decrease of ZEB1 expression after gemcitabine treatment. Remarkably, we detected ZEB1 in 42±3.2%, 11±0.9% and 53±3.5% of cells in vehicle-treated PDXs whereas its expression decreased to 9.2±1.1%, 0.9±0.1% and 33±3.1% after gemcitabine treatment in X-IPC, C-NOR and AO-IPC PDXs, respectively (Figure 3A). ZEB1 frequently colocates with CD44 in vehicle-treated PDXs but rarely in gemcitabine-treated PDXs. The decreased expression of ZEB1 after gemcitabine treatment was confirmed by western blot analysis (Figure 3B and 3C). Furthermore, we evaluated MUC1 expression, which, conversely, is strongly associated with cellular differentiation. As illustrated in Figure 3D, though some cells of the vehicle-treated PDXs expressed MUC1, but never colocalizes with CD44+ cells. However, after gemcitabine treatment we found that the majority of the CD44+ residual cells expressed MUC1 (Figure 3D and F). Western blot analysis confirmed that MUC1 expression dramatically increased in gemcitabine-treated tumors, when compared to vehicle-treated samples (Figure 3E).

Bottom Line: The origin and biological characteristics of residual tumor cells in PDAC still remain unclear.During PDAC relapse, proliferating CD44+ cells decrease expression of ZEB1, while overexpressing the MUC1 protein, and gain morphological and biological characteristics of differentiation.We confirmed the propagation of CD44+ cells in samples from cases of human relapse, following standard PDAC treatment.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche en Cancérologie de Marseille (CRCM), INSERM U1068, CNRS UMR 7258, Aix-Marseille Université and Institut Paoli-Calmettes, Parc Scientifique et Technologique de Luminy, Marseille, France.

ABSTRACT
It has been commonly found that in patients presenting Pancreatic Ductal Adenocarcinoma (PDAC), after a period of satisfactory response to standard treatments, the tumor becomes non-responsive and patient death quickly follows. This phenomenon is mainly due to the rapid and uncontrolled development of the residual tumor. The origin and biological characteristics of residual tumor cells in PDAC still remain unclear. In this work, using PDACs from patients, preserved as xenografts in nude mice, we demonstrated that a residual PDAC tumor originated from a small number of CD44+ cells present in the tumor. During PDAC relapse, proliferating CD44+ cells decrease expression of ZEB1, while overexpressing the MUC1 protein, and gain morphological and biological characteristics of differentiation. Also, we report that CD44+ cells, in primary and residual PDAC tumors, are part of a heterogeneous population, which includes variable numbers of CD133+ and EpCAM+ cells. We confirmed the propagation of CD44+ cells in samples from cases of human relapse, following standard PDAC treatment. Finally, using systemic administration of anti-CD44 antibodies in vivo, we demonstrated that CD44 is an efficient therapeutic target for treating tumor relapse, but not primary PDAC tumors. We conclude that CD44+ cells generate the relapsing tumor and, as such, are themselves promising therapeutic targets for treating patients with recurrent PDAC.

No MeSH data available.


Related in: MedlinePlus