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Aspirin counteracts cancer stem cell features, desmoplasia and gemcitabine resistance in pancreatic cancer.

Zhang Y, Liu L, Fan P, Bauer N, Gladkich J, Ryschich E, Bazhin AV, Giese NA, Strobel O, Hackert T, Hinz U, Gross W, Fortunato F, Herr I - Oncotarget (2015)

Bottom Line: Aspirin significantly induced apoptosis and reduced the viability, self-renewal potential, and expression of proteins involved in inflammation and stem cell signaling.Aspirin also reduced the growth and invasion of tumors in vivo, and it significantly prolonged the survival of mice with orthotopic pancreatic xenografts in combination with gemcitabine.Aspirin showed no obvious toxic effects on normal cells, chick embryos or mice.

View Article: PubMed Central - PubMed

Affiliation: Molecular OncoSurgery, University of Heidelberg and German Cancer Research Center (DKFZ), Heidelberg, Germany.

ABSTRACT
Pancreatic ductal adenocarcinoma (PDA) is characterized by an extremely poor prognosis. An inflammatory microenvironment triggers the pronounced desmoplasia, the selection of cancer stem-like cells (CSCs) and therapy resistance. The anti-inflammatory drug aspirin is suggested to lower the risk for PDA and to improve the treatment, although available results are conflicting and the effect of aspirin to CSC characteristics and desmoplasia in PDA has not yet been investigated. We characterized the influence of aspirin on CSC features, stromal reactions and gemcitabine resistance. Four established and 3 primary PDA cell lines, non-malignant cells, 3 patient tumor-derived CSC-enriched spheroidal cultures and tissues from patients who did or did not receive aspirin before surgery were analyzed using MTT assays, flow cytometry, colony and spheroid formation assays, Western blot analysis, antibody protein arrays, electrophoretic mobility shift assays (EMSAs), immunohistochemistry and in vivo xenotransplantation. Aspirin significantly induced apoptosis and reduced the viability, self-renewal potential, and expression of proteins involved in inflammation and stem cell signaling. Aspirin also reduced the growth and invasion of tumors in vivo, and it significantly prolonged the survival of mice with orthotopic pancreatic xenografts in combination with gemcitabine. This was associated with a decreased expression of markers for progression, inflammation and desmoplasia. These findings were confirmed in tissue samples obtained from patients who had or had not taken aspirin before surgery. Importantly, aspirin sensitized cells that were resistant to gemcitabine and thereby enhanced the therapeutic efficacy. Aspirin showed no obvious toxic effects on normal cells, chick embryos or mice. These results highlight aspirin as an effective, inexpensive and well-tolerated co-treatment to target inflammation, desmoplasia and CSC features PDA.

No MeSH data available.


Related in: MedlinePlus

Aspirin inhibits the development of primary CSC spheroids and enhances gemcitabine efficacy(A) Primary pancreatic CSCs were selected by serial transplantation of freshly resected PDA tumor tissues (T29, T30, T22: compare Table 1) to immunodeficient mice, followed by in vitro culture on ultra-low attachment plates at a low density of 1–2 × 104 cells/mL in serum-free, but growth factor-containing, medium for spheroid formation. (B) One week after culturing in vitro, the spheroids were treated as described. Five days later, the percentage of viable spheroids was determined (1st Gen). The number of spheroids in the control was set to 100%. Representative photographs from T22 spheroids 5 days after treatment at 100× magnification are shown at the right. The bar indicates 100 μm. (C) Cells from treated primary spheroids were collected on microscope slides by cytospin centrifugation. Expression of the proliferation marker Ki67, the apoptosis marker “cleaved, active fragment of caspase-3” (act. Casp3), and the CSC markers c-Met and SOX2 was examined by immunohistochemistry. The number of positive cells was quantified in 10 visual fields at 400× magnification, and the means ± SD are shown in the diagrams (**P < 0.01, *P < 0.05).
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Figure 4: Aspirin inhibits the development of primary CSC spheroids and enhances gemcitabine efficacy(A) Primary pancreatic CSCs were selected by serial transplantation of freshly resected PDA tumor tissues (T29, T30, T22: compare Table 1) to immunodeficient mice, followed by in vitro culture on ultra-low attachment plates at a low density of 1–2 × 104 cells/mL in serum-free, but growth factor-containing, medium for spheroid formation. (B) One week after culturing in vitro, the spheroids were treated as described. Five days later, the percentage of viable spheroids was determined (1st Gen). The number of spheroids in the control was set to 100%. Representative photographs from T22 spheroids 5 days after treatment at 100× magnification are shown at the right. The bar indicates 100 μm. (C) Cells from treated primary spheroids were collected on microscope slides by cytospin centrifugation. Expression of the proliferation marker Ki67, the apoptosis marker “cleaved, active fragment of caspase-3” (act. Casp3), and the CSC markers c-Met and SOX2 was examined by immunohistochemistry. The number of positive cells was quantified in 10 visual fields at 400× magnification, and the means ± SD are shown in the diagrams (**P < 0.01, *P < 0.05).

Mentions: To extend these data, we used human primary CSC marker-enriched spheroidal-growing PDA cells isolated from resected PDA tissues from 3 different patients (T22, T29, and T30) (Table 1) followed by serial transplantation to mice (Figure 4A). At passage 6–12, the tumor cells were isolated from xenografts and cultured anchorage-independently in vitro, as we described previously [22, 29]. During this procedure, the highly aggressive cancer cells are enriched by clonal selection, which was confirmed by the enrichment of the CSC markers CD133 and c-Met from below 20% in the primary tumor to about 80% in the spheroidal cultures [22, 29]. The rational for this procedure was that a similar selection occurs in patient tumors: At the beginning of the disease most of tumors respond to chemotherapy, but after several cycles, the tumor acquires resistance, and this may be due to the clonal selection of the highly resistant cancer cells. The isolated primary CSCs were exposed to aspirin, gemcitabine, or both in combination. Five days later, the number of viable spheroids from the first generation (1st Gen) was evaluated, followed by a second round of treatment (2nd Gen). Whereas aspirin and gemcitabine alone reduced the percentage and size of the spheroids, combination treatment was significantly more effective and almost completely prevented the generation of secondary spheroids (Figure 4B). To evaluate the underlying signaling events, primary CSCs were collected on a microscope slide following cytospin centrifugation 72 h after treatment and then examined by immunohistochemistry and counting the percentage of positive cells. Aspirin and gemcitabine led to a significant reduction of proliferation, as indicated by labeling with Ki67-specific antibodies, expression of c-Met and SOX-2, and induction of the active form of caspase-3, indicating the induction of apoptosis (Figure 4C, Figure S3). These effects were observed with all treatments but were most significant for aspirin and gemcitabine in combination.


Aspirin counteracts cancer stem cell features, desmoplasia and gemcitabine resistance in pancreatic cancer.

Zhang Y, Liu L, Fan P, Bauer N, Gladkich J, Ryschich E, Bazhin AV, Giese NA, Strobel O, Hackert T, Hinz U, Gross W, Fortunato F, Herr I - Oncotarget (2015)

Aspirin inhibits the development of primary CSC spheroids and enhances gemcitabine efficacy(A) Primary pancreatic CSCs were selected by serial transplantation of freshly resected PDA tumor tissues (T29, T30, T22: compare Table 1) to immunodeficient mice, followed by in vitro culture on ultra-low attachment plates at a low density of 1–2 × 104 cells/mL in serum-free, but growth factor-containing, medium for spheroid formation. (B) One week after culturing in vitro, the spheroids were treated as described. Five days later, the percentage of viable spheroids was determined (1st Gen). The number of spheroids in the control was set to 100%. Representative photographs from T22 spheroids 5 days after treatment at 100× magnification are shown at the right. The bar indicates 100 μm. (C) Cells from treated primary spheroids were collected on microscope slides by cytospin centrifugation. Expression of the proliferation marker Ki67, the apoptosis marker “cleaved, active fragment of caspase-3” (act. Casp3), and the CSC markers c-Met and SOX2 was examined by immunohistochemistry. The number of positive cells was quantified in 10 visual fields at 400× magnification, and the means ± SD are shown in the diagrams (**P < 0.01, *P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4496413&req=5

Figure 4: Aspirin inhibits the development of primary CSC spheroids and enhances gemcitabine efficacy(A) Primary pancreatic CSCs were selected by serial transplantation of freshly resected PDA tumor tissues (T29, T30, T22: compare Table 1) to immunodeficient mice, followed by in vitro culture on ultra-low attachment plates at a low density of 1–2 × 104 cells/mL in serum-free, but growth factor-containing, medium for spheroid formation. (B) One week after culturing in vitro, the spheroids were treated as described. Five days later, the percentage of viable spheroids was determined (1st Gen). The number of spheroids in the control was set to 100%. Representative photographs from T22 spheroids 5 days after treatment at 100× magnification are shown at the right. The bar indicates 100 μm. (C) Cells from treated primary spheroids were collected on microscope slides by cytospin centrifugation. Expression of the proliferation marker Ki67, the apoptosis marker “cleaved, active fragment of caspase-3” (act. Casp3), and the CSC markers c-Met and SOX2 was examined by immunohistochemistry. The number of positive cells was quantified in 10 visual fields at 400× magnification, and the means ± SD are shown in the diagrams (**P < 0.01, *P < 0.05).
Mentions: To extend these data, we used human primary CSC marker-enriched spheroidal-growing PDA cells isolated from resected PDA tissues from 3 different patients (T22, T29, and T30) (Table 1) followed by serial transplantation to mice (Figure 4A). At passage 6–12, the tumor cells were isolated from xenografts and cultured anchorage-independently in vitro, as we described previously [22, 29]. During this procedure, the highly aggressive cancer cells are enriched by clonal selection, which was confirmed by the enrichment of the CSC markers CD133 and c-Met from below 20% in the primary tumor to about 80% in the spheroidal cultures [22, 29]. The rational for this procedure was that a similar selection occurs in patient tumors: At the beginning of the disease most of tumors respond to chemotherapy, but after several cycles, the tumor acquires resistance, and this may be due to the clonal selection of the highly resistant cancer cells. The isolated primary CSCs were exposed to aspirin, gemcitabine, or both in combination. Five days later, the number of viable spheroids from the first generation (1st Gen) was evaluated, followed by a second round of treatment (2nd Gen). Whereas aspirin and gemcitabine alone reduced the percentage and size of the spheroids, combination treatment was significantly more effective and almost completely prevented the generation of secondary spheroids (Figure 4B). To evaluate the underlying signaling events, primary CSCs were collected on a microscope slide following cytospin centrifugation 72 h after treatment and then examined by immunohistochemistry and counting the percentage of positive cells. Aspirin and gemcitabine led to a significant reduction of proliferation, as indicated by labeling with Ki67-specific antibodies, expression of c-Met and SOX-2, and induction of the active form of caspase-3, indicating the induction of apoptosis (Figure 4C, Figure S3). These effects were observed with all treatments but were most significant for aspirin and gemcitabine in combination.

Bottom Line: Aspirin significantly induced apoptosis and reduced the viability, self-renewal potential, and expression of proteins involved in inflammation and stem cell signaling.Aspirin also reduced the growth and invasion of tumors in vivo, and it significantly prolonged the survival of mice with orthotopic pancreatic xenografts in combination with gemcitabine.Aspirin showed no obvious toxic effects on normal cells, chick embryos or mice.

View Article: PubMed Central - PubMed

Affiliation: Molecular OncoSurgery, University of Heidelberg and German Cancer Research Center (DKFZ), Heidelberg, Germany.

ABSTRACT
Pancreatic ductal adenocarcinoma (PDA) is characterized by an extremely poor prognosis. An inflammatory microenvironment triggers the pronounced desmoplasia, the selection of cancer stem-like cells (CSCs) and therapy resistance. The anti-inflammatory drug aspirin is suggested to lower the risk for PDA and to improve the treatment, although available results are conflicting and the effect of aspirin to CSC characteristics and desmoplasia in PDA has not yet been investigated. We characterized the influence of aspirin on CSC features, stromal reactions and gemcitabine resistance. Four established and 3 primary PDA cell lines, non-malignant cells, 3 patient tumor-derived CSC-enriched spheroidal cultures and tissues from patients who did or did not receive aspirin before surgery were analyzed using MTT assays, flow cytometry, colony and spheroid formation assays, Western blot analysis, antibody protein arrays, electrophoretic mobility shift assays (EMSAs), immunohistochemistry and in vivo xenotransplantation. Aspirin significantly induced apoptosis and reduced the viability, self-renewal potential, and expression of proteins involved in inflammation and stem cell signaling. Aspirin also reduced the growth and invasion of tumors in vivo, and it significantly prolonged the survival of mice with orthotopic pancreatic xenografts in combination with gemcitabine. This was associated with a decreased expression of markers for progression, inflammation and desmoplasia. These findings were confirmed in tissue samples obtained from patients who had or had not taken aspirin before surgery. Importantly, aspirin sensitized cells that were resistant to gemcitabine and thereby enhanced the therapeutic efficacy. Aspirin showed no obvious toxic effects on normal cells, chick embryos or mice. These results highlight aspirin as an effective, inexpensive and well-tolerated co-treatment to target inflammation, desmoplasia and CSC features PDA.

No MeSH data available.


Related in: MedlinePlus