Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death methods
Aim. Evidence-backed execution summary for Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death methods from Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death.
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mouse
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Immunogenicity of caspase-dependent, DX-induced cell death
reagent used in the protocol.
- Use
- As shown above, DX induced immunogenic tumor cell death. However, concomitant treatment with the broad-spectrum caspase inhibitor Z-VAD-fmk (DXZ) abolished the immunogenic potential of the treatment with DX ( ). Similar inhibitory effects were obtained when Z-VAD-fmk was replaced by a chemically related caspase inhi...
Immunogenicity of caspase-dependent, DX-induced cell death
reagent used in the protocol.
- Use
- Inhibition of immunogenicity by the caspase inhibitor p35. (A) FACS analysis of CT26 cells transfected with vector only (Neo) or p35 cultured alone or in the presence of DX as in A. Numbers indicate the percentage of cells (X ± SD, n = 5) in each quadrant. (B) Clonogenic survival of Neo or p35-transfected cells...
Critical role of DCs in the immune response elicited by dying tumor cells
reagent used in the protocol.
- Use
- Contribution of specific CTLs and DCs to the immune response against DX-treated tumor cells. (A) Percentages of CD8 + lymph node cells expressing TCR that interact with the OVA-derived peptide SIINFEKL presented by H-2K b 5 d after challenge with either peptide plus adjuvant (P+A) or with DX, DX F/T (cells treated w...
Ex vivo and in vivo induction of immunogenic apoptosis
reagent used in the protocol.
- Use
- We investigated whether it would be required to immunize animals with dying cells cultured as an established cell line or whether it would be feasible to take advantage of resected tumors (as this would be the case in human cancer patients). CT26 tumors were generated in BALB/c mice and then surgically removed and e...
MATERIALS AND METHODS
reagent used in the protocol.
- Use
- CT26, PROb, B16F10, B16/F10.9-OVA, and B16F10A2/gp100 (B16F10 transfected with gp100 and HLA A2.1 and selected in 50 µg/ml hygromicin and G418) cells were cultured at 37°C under 5% CO 2 in RPMI 1640 medium supplemented with 10% FCS, penicillin, streptomycin, 1 mM pyruvate, and 10 mM Hepes in the presence o...
Cell death assays.
reagent used in the protocol.
- Use
- Cells were trypsinized and subjected to cytofluorometric analysis with a FACSVantage after staining with 2.5 µM DAPI for 10 min (Invitrogen) for determination of cell viability, and annexin V was conjugated with fluorescein isothiocyanate (Bender Medsystems) for the assessment of phosphatidylserine exposure (,...
Immunoblot analyses.
reagent used in the protocol.
- Use
- Cells were washed with cold PBS at 4°C and lysed in a buffer containing 50 mM Tris HCl, pH 6.8, 10% glycerol, and 2% SDS. Primary antibodies detecting activated caspase 3 (dilution 1/1,000; Cell Signaling Technology), HSP70 (dilution 1/1,000; Stratagene) or 2 µg/ml HMGB-1 (BD Biosciences) were revealed wit...
Antitumor vaccination and assessment of tumor growth in vivo.
reagent used in the protocol.
- Use
- All animals were maintained in specific pathogen-free conditions, and all experiments followed the Federation of European Laboratory Animal Science Association guidelines. 3 × 10 6 treated CT26 cells were inoculated subcutaneously in 200 µl PBS into 6-wk-old female BALB/c mice (Charles River Laboratories)...
Immunogenicity of caspase-dependent, DX-induced cell death
As shown above, DX induced immunogenic tumor cell death. However, concomitant treatment with the broad-spectrum caspase inhibitor Z-VAD-fmk (DXZ) abolished the immunogenic potential of the treatment with DX ( ). Similar inhibitory effects were obtained when Z-VAD-fmk was replaced by a chemically related caspase inhi...
- Use
- As shown above, DX induced immunogenic tumor cell death. However, concomitant treatment with the broad-spectrum caspase inhibitor Z-VAD-fmk (DXZ) abolished the immunogenic potential of the treatment with DX ( ). Similar inhibitory effects were obtained when Z-VAD-fmk was replaced by a chemically related caspase inhi...
Immunogenicity of caspase-dependent, DX-induced cell death
Inhibition of immunogenicity by the caspase inhibitor p35. (A) FACS analysis of CT26 cells transfected with vector only (Neo) or p35 cultured alone or in the presence of DX as in A. Numbers indicate the percentage of cells (X ± SD, n = 5) in each quadrant. (B) Clonogenic survival of Neo or p35-transfected cells...
- Use
- Inhibition of immunogenicity by the caspase inhibitor p35. (A) FACS analysis of CT26 cells transfected with vector only (Neo) or p35 cultured alone or in the presence of DX as in A. Numbers indicate the percentage of cells (X ± SD, n = 5) in each quadrant. (B) Clonogenic survival of Neo or p35-transfected cells...
Critical role of DCs in the immune response elicited by dying tumor cells
In an attempt to understand why DX-induced cell death is immunogenic, we challenged DCs with dead or dying cells and measured their response. DX-treated CT26 cells were phagocytosed by all CD11c + /IA d+ DC subpopulations (CD11b + B220 + DCs in A and CD8α + DCs not depicted) present in the spleen, whereas nonim...
- Use
- In an attempt to understand why DX-induced cell death is immunogenic, we challenged DCs with dead or dying cells and measured their response. DX-treated CT26 cells were phagocytosed by all CD11c + /IA d+ DC subpopulations (CD11b + B220 + DCs in A and CD8α + DCs not depicted) present in the spleen, whereas nonim...
Critical role of DCs in the immune response elicited by dying tumor cells
Effect of dying tumor cells on DCs. (A and B) In vitro phagocytosis of the DX-, DXZ-, or MC-treated cells (stained with CMTMR) by spleen DCs from Flt3L-injected mice. Representative FACS diagrams are depicted in A, and confocal images of dying tumor cells (red) phagocytosed by purified DC subsets (green) are shown i...
- Use
- Effect of dying tumor cells on DCs. (A and B) In vitro phagocytosis of the DX-, DXZ-, or MC-treated cells (stained with CMTMR) by spleen DCs from Flt3L-injected mice. Representative FACS diagrams are depicted in A, and confocal images of dying tumor cells (red) phagocytosed by purified DC subsets (green) are shown i...
Critical role of DCs in the immune response elicited by dying tumor cells
Contribution of specific CTLs and DCs to the immune response against DX-treated tumor cells. (A) Percentages of CD8 + lymph node cells expressing TCR that interact with the OVA-derived peptide SIINFEKL presented by H-2K b 5 d after challenge with either peptide plus adjuvant (P+A) or with DX, DX F/T (cells treated w...
- Use
- Contribution of specific CTLs and DCs to the immune response against DX-treated tumor cells. (A) Percentages of CD8 + lymph node cells expressing TCR that interact with the OVA-derived peptide SIINFEKL presented by H-2K b 5 d after challenge with either peptide plus adjuvant (P+A) or with DX, DX F/T (cells treated w...
DX-elicited apoptosis is immunogenic in several tumor models
One single subcutaneous injection of DX-treated CT26 cells conferred a strong protection against the development of pulmonary metastases induced by simultaneous intravenous injection of tumor cells ( A). In this system, MC-treated and F/T cells were inefficient. Freeze-thawing of DX-treated CT26 cells (DX F/T) annih...
- Use
- One single subcutaneous injection of DX-treated CT26 cells conferred a strong protection against the development of pulmonary metastases induced by simultaneous intravenous injection of tumor cells ( A). In this system, MC-treated and F/T cells were inefficient. Freeze-thawing of DX-treated CT26 cells (DX F/T) annih...
Ex vivo and in vivo induction of immunogenic apoptosis
We investigated whether it would be required to immunize animals with dying cells cultured as an established cell line or whether it would be feasible to take advantage of resected tumors (as this would be the case in human cancer patients). CT26 tumors were generated in BALB/c mice and then surgically removed and e...
- Use
- We investigated whether it would be required to immunize animals with dying cells cultured as an established cell line or whether it would be feasible to take advantage of resected tumors (as this would be the case in human cancer patients). CT26 tumors were generated in BALB/c mice and then surgically removed and e...
MATERIALS AND METHODS
CT26, PROb, B16F10, B16/F10.9-OVA, and B16F10A2/gp100 (B16F10 transfected with gp100 and HLA A2.1 and selected in 50 µg/ml hygromicin and G418) cells were cultured at 37°C under 5% CO 2 in RPMI 1640 medium supplemented with 10% FCS, penicillin, streptomycin, 1 mM pyruvate, and 10 mM Hepes in the presence o...
- Use
- CT26, PROb, B16F10, B16/F10.9-OVA, and B16F10A2/gp100 (B16F10 transfected with gp100 and HLA A2.1 and selected in 50 µg/ml hygromicin and G418) cells were cultured at 37°C under 5% CO 2 in RPMI 1640 medium supplemented with 10% FCS, penicillin, streptomycin, 1 mM pyruvate, and 10 mM Hepes in the presence o...
Statistical analyses.
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Data are presented as arithmetic means ± SD or percentages. All statistical analyses were performed using JMP software (SAS Institute Inc.). The Student's t test was used to compare continuous variables (comparison of tumor growth), the Chi square test was used for nonparametrical variables (comparison of anima...
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Critical role of DCs in the immune response elicited by dying tumor cells
Effect of dying tumor cells on DCs. (A and B) In vitro phagocytosis of the DX-, DXZ-, or MC-treated cells (stained with CMTMR) by spleen DCs from Flt3L-injected mice. Representative FACS diagrams are depicted in A, and confocal images of dying tumor cells (red) phagocytosed by purified DC subsets (green) are shown in B. (C) DC maturation of splenic DCs induced by LPS (positive control) and dying or dead CT26 cells. Percentage values in A and C are means of three independent determinations ± SD. (D) Failure of Z-VAD-fmk to inhibit the phagocytosis of DX-treated CT26 cells by DCs. DCs generated as in D were incubated for 90 min with a twofold excess of DX-treated CT26 cells in the presence of the indicated concentrations of Z-VAD-fmk, and the percentage of DCs containing dying or dead CT26 cells was determined as in A. (E) Failure of DX to activate DCs. Bone marrow-derived DC...
Critical role of DCs in the immune response elicited by dying tumor cells
Contribution of specific CTLs and DCs to the immune response against DX-treated tumor cells. (A) Percentages of CD8 + lymph node cells expressing TCR that interact with the OVA-derived peptide SIINFEKL presented by H-2K b 5 d after challenge with either peptide plus adjuvant (P+A) or with DX, DX F/T (cells treated with DX and then F/T), DXZ-, MC-, and F/T-treated B16-OVA cells (three experiments). (B) Absolute number of specific CD8 + cells per lymph node determined as in A. Values are X ± SD ( n = 3). (C) IFN-γ production as measured after in vitro culture with SIINFEKL in the same experiment. Control values are <50 pg/ml. (D) Effect of the depletion of DCs on the accumulation of specific T cells. Transgenic mice specifically expressing the diphteria toxin (DT) receptor in DCs were pretreated with PBS alone or a dose of diphteria toxin that depletes DCs. The mice were then...
Ex vivo and in vivo induction of immunogenic apoptosis
We investigated whether it would be required to immunize animals with dying cells cultured as an established cell line or whether it would be feasible to take advantage of resected tumors (as this would be the case in human cancer patients). CT26 tumors were generated in BALB/c mice and then surgically removed and enzymatically dissociated optionally after a step of cryostorage in DMSO-containing medium, treated with DX in vitro, and used as antitumor vaccine ( A). This ex vivo removal/in vitro treatment elicited an efficient antitumor immune response if DX was added during the in vitro incubation, although cryostored cells were less efficient than freshly recovered tumor cells. The omission of DX curtailed the efficacy of the vaccine ( A). As an alternative to this ex vivo/in vitro protocol of vaccine generation, we injected DX into palpable (>125 mm 3 ) subcutaneous CT26 tumors esta...
MATERIALS AND METHODS
CT26, PROb, B16F10, B16/F10.9-OVA, and B16F10A2/gp100 (B16F10 transfected with gp100 and HLA A2.1 and selected in 50 µg/ml hygromicin and G418) cells were cultured at 37°C under 5% CO 2 in RPMI 1640 medium supplemented with 10% FCS, penicillin, streptomycin, 1 mM pyruvate, and 10 mM Hepes in the presence of DX for 24 h (25 µM for CT26, 30 µM for PROb, and 2.5 µM for B16F10 and its derivatives), 5 µM daunorubicin for 24 h (GE Healthcare), 1 µM idarubicin for 24 h (Aventis), 30 µM MC for 48 h (Sanofi-Synthelabo), and/or 100 µM zVAD-fmk for 24 h (Bachem). Necrosis was induced by one freeze-thaw cycle in liquid N 2 and a 37°C water bath. CT26 cells were stably transfected with vector only (Neo) or with a pcDNA3.1 vector encoding p35 (, ). In one experiment, 100 µM DX was added for 15 min to cells cultured in MC for 2 d. The incorpora...
Cell death assays.
Cells were trypsinized and subjected to cytofluorometric analysis with a FACSVantage after staining with 2.5 µM DAPI for 10 min (Invitrogen) for determination of cell viability, and annexin V was conjugated with fluorescein isothiocyanate (Bender Medsystems) for the assessment of phosphatidylserine exposure (, ). TUNEL assays were performed on cells (let to adhere for 1 h in PBS on polylysine-coated slides [O. Kindler GmbH] and fixed with 4% paraformaldehyde for 30 min) using the In situ Cell Death Detection Kit (Roche) and a fluorescence microscope (IRE2; Leica) equipped with a camera (DC300F; Leica). For electron microscopy, cells (fixed for 1 h at 4°C in 2.5% glutaraldehyde in phosphate buffer, pH 7.4, washed, and fixed again in 2% osmium tetroxide and then embedded in Epon) and ultrathin sections (80 nm) were stained with uranyl acetate and lead citrate and examined wit...
Immunoblot analyses.
Cells were washed with cold PBS at 4°C and lysed in a buffer containing 50 mM Tris HCl, pH 6.8, 10% glycerol, and 2% SDS. Primary antibodies detecting activated caspase 3 (dilution 1/1,000; Cell Signaling Technology), HSP70 (dilution 1/1,000; Stratagene) or 2 µg/ml HMGB-1 (BD Biosciences) were revealed with the appropriate horseradish peroxidase-labeled secondary antibody (Southern Biotechnology Associates, Inc.) and detected by enhanced chemiluminescence (Pierce Chemical Co.). Anti-actin or anti-GAPDH (Chemicon) was used to control equal loading.
Antitumor vaccination and assessment of tumor growth in vivo.
All animals were maintained in specific pathogen-free conditions, and all experiments followed the Federation of European Laboratory Animal Science Association guidelines. 3 × 10 6 treated CT26 cells were inoculated subcutaneously in 200 µl PBS into 6-wk-old female BALB/c mice (Charles River Laboratories) into the lower flank, whereas 5 × 10 5 untreated control cells were inoculated into the contralateral flank ( ). PROb cells (3 × 10 6 treated cells and 10 6 control cells) were injected subcutaneously into the lower flanks of syngeneic 2-mo-old BDIX rats (Charles River Laboratories), and B16A2 and B16F10 (1.8 × 10 5 treated cells and 3 × 10 4 control cells) were injected into 6-wk-old female HHD2 transgenic mice (bred in the Institut Gustave Roussy [IGR] animal facility) and C57BL/6 mice (Charles River Laboratories), respectively. For the tumorigenicity...
Assessment of specific T cell responses.
The CTL response against the immunodominant CT26-specific AH1 H2L d -restricted peptide (SPSYVYHQF; Neosystem; reference ) was determined. Splenocytes were isolated from immunized mice 10 d after injection of apoptotic cells and restimulated in vitro for 5 d with or without 5 µg/ml AH1 in the presence of syngeneic irradiated naive spleen cells. The cytotoxic activity was determined during a classic 5-h in vitro 51 Cr-release assay using 51 Cr-P815 tumor cells loaded with 50 µM AH1 peptide as target cells ( ). The percentage of specific lysis was calculated as 100 × (experimental release - spontaneous release)/(maximal release - spontaneous release). Maximum release was obtained by adding 10% Triton X-405 to target cells, and spontaneous release was determined by incubating target cells in medium alone. 5-6-wk-old female C57BL/6 mice (H-2 b ) were injec...
Measurement outputs
What raw and processed outputs should exist?
Inhibition of immunogenicity by the caspase inhibitor p35. (A) FACS analysis of CT26 cells transfected with vector only (Neo) or p35 cultured alone or in the presence of DX as i...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
In an attempt to understand why DX-induced cell death is immunogenic, we challenged DCs with dead or dying cells and measured their response. DX-treated CT26 cells were phagocyt...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Effect of dying tumor cells on DCs. (A and B) In vitro phagocytosis of the DX-, DXZ-, or MC-treated cells (stained with CMTMR) by spleen DCs from Flt3L-injected mice. Representa...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Cells were trypsinized and subjected to cytofluorometric analysis with a FACSVantage after staining with 2.5 µM DAPI for 10 min (Invitrogen) for determination of cell viabi...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect the raw assay or blot output and retain identifiers for each sample and experimental group.
inferred from protocolPreprocessing / cleaning
One single subcutaneous injection of DX-treated CT26 cells conferred a strong protection against the development of pulmonary metastases induced by simultaneous intravenous injection of tumor cells ( A).
from paperScoring or quantification
Quantify the primary readouts for this experiment: Inhibition of immunogenicity by the caspase inhibitor p35. (A) FACS analysis of CT26 cells transfected with vector only (Neo) or p35 cultured alone or in the presence of DX as i...; In an attempt to understand why DX-induced cell death is immunogenic, we challenged DCs with dead or dying cells and measured their response. DX-treated CT26 cells were phagocyt...; Effect of dying tumor cells on DCs. (A and B) In vitro phagocytosis of the DX-, DXZ-, or MC-treated cells (stained with CMTMR) by spleen DCs from Flt3L-injected mice. Representa...; Cells were trypsinized and subjected to cytofluorometric analysis with a FACSVantage after staining with 2.5 µM DAPI for 10 min (Invitrogen) for determination of cell viabi....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
One single subcutaneous injection of DX-treated CT26 cells conferred a strong protection against the development of pulmonary metastases induced by simultaneous intravenous inje...; Data are presented as arithmetic means ± SD or percentages. All statistical analyses were performed using JMP software (SAS Institute Inc.). The Student's t test was used t...
from paperReporting output
Report representative outputs alongside summary comparisons for Inhibition of immunogenicity by the caspase inhibitor p35. (A) FACS analysis of CT26 cells transfected with vector only (Neo) or p35 cultured alone or in the presence of DX as i..., In an attempt to understand why DX-induced cell death is immunogenic, we challenged DCs with dead or dying cells and measured their response. DX-treated CT26 cells were phagocyt..., Effect of dying tumor cells on DCs. (A and B) In vitro phagocytosis of the DX-, DXZ-, or MC-treated cells (stained with CMTMR) by spleen DCs from Flt3L-injected mice. Representa..., Cells were trypsinized and subjected to cytofluorometric analysis with a FACSVantage after staining with 2.5 µM DAPI for 10 min (Invitrogen) for determination of cell viabi....
inferred from protocolStructured statistical methods
One single subcutaneous injection of DX-treated CT26 cells conferred a strong protection against the development of pulmonary metastases induced by simultaneous intravenous inje...; Data are presented as arithmetic means ± SD or percentages. All statistical analyses were performed using JMP software (SAS Institute Inc.). The Student's t test was used t...
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Evidence quotes (8)
Effect of dying tumor cells on DCs. (A and B) In vitro phagocytosis of the DX-, DXZ-, or MC-treated cells (stained with CMTMR) by spleen DCs from Flt3L-injected mice. Representative FACS diagrams are depicted in A, and confocal images of dying tumor cells (red) phagocytosed by purified DC subsets (green) are shown in B. (C) DC maturation of splenic DCs induced by LPS (positive control) and dying or dead CT26 cells. Percentage values in A and C are means of three independent determinations ± SD. (D) Failure of Z-VAD-fmk to inhibit the phagocytosis of DX-treated CT26 cells by DCs. DCs generated as in D were incubated for 90 min with a twofold excess of DX-treated CT26 cells in the presence of the indicated concentrations of Z-VAD-fmk, and the percentage of DCs containing dying or dead CT26 cells was determined as in A. (E) Failure of DX to activate DCs. Bone marrow-derived DCs were activated with LPS as a positive control or with the indicated concentration of DX, and the frequency of apoptotic cells (annexin V + ) and CD86 + cells was determined by immunofluorescence and cytofluorometric analysis. Percentage values in A and C-E are means of three independent dete...
Contribution of specific CTLs and DCs to the immune response against DX-treated tumor cells. (A) Percentages of CD8 + lymph node cells expressing TCR that interact with the OVA-derived peptide SIINFEKL presented by H-2K b 5 d after challenge with either peptide plus adjuvant (P+A) or with DX, DX F/T (cells treated with DX and then F/T), DXZ-, MC-, and F/T-treated B16-OVA cells (three experiments). (B) Absolute number of specific CD8 + cells per lymph node determined as in A. Values are X ± SD ( n = 3). (C) IFN-γ production as measured after in vitro culture with SIINFEKL in the same experiment. Control values are <50 pg/ml. (D) Effect of the depletion of DCs on the accumulation of specific T cells. Transgenic mice specifically expressing the diphteria toxin (DT) receptor in DCs were pretreated with PBS alone or a dose of diphteria toxin that depletes DCs. The mice were then challenged with DX-treated B16-OVA cells into the food pad. Draining lymph nodes were recovered 48 h later and stained for simultaneous detection of CD8 and OVA peptide-specific T cell receptors. Representative FACS pictograms are shown and values are X ± SD ( n = 3).
We investigated whether it would be required to immunize animals with dying cells cultured as an established cell line or whether it would be feasible to take advantage of resected tumors (as this would be the case in human cancer patients). CT26 tumors were generated in BALB/c mice and then surgically removed and enzymatically dissociated optionally after a step of cryostorage in DMSO-containing medium, treated with DX in vitro, and used as antitumor vaccine ( A). This ex vivo removal/in vitro treatment elicited an efficient antitumor immune response if DX was added during the in vitro incubation, although cryostored cells were less efficient than freshly recovered tumor cells. The omission of DX curtailed the efficacy of the vaccine ( A). As an alternative to this ex vivo/in vitro protocol of vaccine generation, we injected DX into palpable (>125 mm 3 ) subcutaneous CT26 tumors established in BALB/c mice. Using this protocol of intratumoral as opposed to intravenous (not depicted) chemotherapy, we achieved stable disease or complete tumor regression in 16 out of 40 (40%) of the animals ( B). We have no explanation why some animals showed no therapeutic effect, others demonstra...
CT26, PROb, B16F10, B16/F10.9-OVA, and B16F10A2/gp100 (B16F10 transfected with gp100 and HLA A2.1 and selected in 50 µg/ml hygromicin and G418) cells were cultured at 37°C under 5% CO 2 in RPMI 1640 medium supplemented with 10% FCS, penicillin, streptomycin, 1 mM pyruvate, and 10 mM Hepes in the presence of DX for 24 h (25 µM for CT26, 30 µM for PROb, and 2.5 µM for B16F10 and its derivatives), 5 µM daunorubicin for 24 h (GE Healthcare), 1 µM idarubicin for 24 h (Aventis), 30 µM MC for 48 h (Sanofi-Synthelabo), and/or 100 µM zVAD-fmk for 24 h (Bachem). Necrosis was induced by one freeze-thaw cycle in liquid N 2 and a 37°C water bath. CT26 cells were stably transfected with vector only (Neo) or with a pcDNA3.1 vector encoding p35 (, ). In one experiment, 100 µM DX was added for 15 min to cells cultured in MC for 2 d. The incorporation of DX into washed (three times in PBS) cells was measured by assessing the DX-specific red fluorescence in a FACSVantage (Becton Dickinson) equipped with an argon laser.
Cells were trypsinized and subjected to cytofluorometric analysis with a FACSVantage after staining with 2.5 µM DAPI for 10 min (Invitrogen) for determination of cell viability, and annexin V was conjugated with fluorescein isothiocyanate (Bender Medsystems) for the assessment of phosphatidylserine exposure (, ). TUNEL assays were performed on cells (let to adhere for 1 h in PBS on polylysine-coated slides [O. Kindler GmbH] and fixed with 4% paraformaldehyde for 30 min) using the In situ Cell Death Detection Kit (Roche) and a fluorescence microscope (IRE2; Leica) equipped with a camera (DC300F; Leica). For electron microscopy, cells (fixed for 1 h at 4°C in 2.5% glutaraldehyde in phosphate buffer, pH 7.4, washed, and fixed again in 2% osmium tetroxide and then embedded in Epon) and ultrathin sections (80 nm) were stained with uranyl acetate and lead citrate and examined with an electron microscope (902; Leo) at 80 kV.
Cells were washed with cold PBS at 4°C and lysed in a buffer containing 50 mM Tris HCl, pH 6.8, 10% glycerol, and 2% SDS. Primary antibodies detecting activated caspase 3 (dilution 1/1,000; Cell Signaling Technology), HSP70 (dilution 1/1,000; Stratagene) or 2 µg/ml HMGB-1 (BD Biosciences) were revealed with the appropriate horseradish peroxidase-labeled secondary antibody (Southern Biotechnology Associates, Inc.) and detected by enhanced chemiluminescence (Pierce Chemical Co.). Anti-actin or anti-GAPDH (Chemicon) was used to control equal loading.
All animals were maintained in specific pathogen-free conditions, and all experiments followed the Federation of European Laboratory Animal Science Association guidelines. 3 × 10 6 treated CT26 cells were inoculated subcutaneously in 200 µl PBS into 6-wk-old female BALB/c mice (Charles River Laboratories) into the lower flank, whereas 5 × 10 5 untreated control cells were inoculated into the contralateral flank ( ). PROb cells (3 × 10 6 treated cells and 10 6 control cells) were injected subcutaneously into the lower flanks of syngeneic 2-mo-old BDIX rats (Charles River Laboratories), and B16A2 and B16F10 (1.8 × 10 5 treated cells and 3 × 10 4 control cells) were injected into 6-wk-old female HHD2 transgenic mice (bred in the Institut Gustave Roussy [IGR] animal facility) and C57BL/6 mice (Charles River Laboratories), respectively. For the tumorigenicity assay, 3 × 10 6 treated or untreated CT26 cells were injected subcutaneously into nu/nu mice (IGR animal facility). To assess the specificity of the immune response against CT26, we injected either 5 × 10 5 or 5 × 10 6 of CT26 (for the mice immunized in a standard protocol or vaccinat...
The CTL response against the immunodominant CT26-specific AH1 H2L d -restricted peptide (SPSYVYHQF; Neosystem; reference ) was determined. Splenocytes were isolated from immunized mice 10 d after injection of apoptotic cells and restimulated in vitro for 5 d with or without 5 µg/ml AH1 in the presence of syngeneic irradiated naive spleen cells. The cytotoxic activity was determined during a classic 5-h in vitro 51 Cr-release assay using 51 Cr-P815 tumor cells loaded with 50 µM AH1 peptide as target cells ( ). The percentage of specific lysis was calculated as 100 × (experimental release - spontaneous release)/(maximal release - spontaneous release). Maximum release was obtained by adding 10% Triton X-405 to target cells, and spontaneous release was determined by incubating target cells in medium alone. 5-6-wk-old female C57BL/6 mice (H-2 b ) were injected into both footpads with PBS and 50 µg OVA peptide (H-2K b -restricted, SIINFEKL, amino acids 257-264; Eurogentec) mixed with incomplete Freund adjuvant (Sigma-Aldrich) or 10 6 B16/F10.9-OVA cells. Animals were killed 5 d after footpad immunization, and popliteal and inguinal lymph nod...
Machine-readable layer
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"name": "Critical role of DCs in the immune response elicited by dying tumor cells",
"text": "Effect of dying tumor cells on DCs. (A and B) In vitro phagocytosis of the DX-, DXZ-, or MC-treated cells (stained with CMTMR) by spleen DCs from Flt3L-injected mice. Representative FACS diagrams are depicted in A, and confocal images of dying tumor cells (red) phagocytosed by purified DC subsets (green) are shown in B. (C) DC maturation of splenic DCs induced by LPS (positive control) and dying or dead CT26 cells. Percentage values in A and C are means of three independent determinations ± SD. (D) Failure of Z-VAD-fmk to inhibit the phagocytosis of DX-treated CT26 cells by DCs. DCs generated as in D were incubated for 90 min with a twofold excess of DX-treated CT26 cells in the presence of the indicated concentrations of Z-VAD-fmk, and the percentage of DCs containing dying or dead CT26 cells was determined as in A. (E) Failure of DX to activate DCs. Bone marrow-derived DC..."
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"name": "Critical role of DCs in the immune response elicited by dying tumor cells",
"text": "Contribution of specific CTLs and DCs to the immune response against DX-treated tumor cells. (A) Percentages of CD8 + lymph node cells expressing TCR that interact with the OVA-derived peptide SIINFEKL presented by H-2K b 5 d after challenge with either peptide plus adjuvant (P+A) or with DX, DX F/T (cells treated with DX and then F/T), DXZ-, MC-, and F/T-treated B16-OVA cells (three experiments). (B) Absolute number of specific CD8 + cells per lymph node determined as in A. Values are X ± SD ( n = 3). (C) IFN-γ production as measured after in vitro culture with SIINFEKL in the same experiment. Control values are <50 pg/ml. (D) Effect of the depletion of DCs on the accumulation of specific T cells. Transgenic mice specifically expressing the diphteria toxin (DT) receptor in DCs were pretreated with PBS alone or a dose of diphteria toxin that depletes DCs. The mice were then..."
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"name": "Ex vivo and in vivo induction of immunogenic apoptosis",
"text": "We investigated whether it would be required to immunize animals with dying cells cultured as an established cell line or whether it would be feasible to take advantage of resected tumors (as this would be the case in human cancer patients). CT26 tumors were generated in BALB/c mice and then surgically removed and enzymatically dissociated optionally after a step of cryostorage in DMSO-containing medium, treated with DX in vitro, and used as antitumor vaccine ( A). This ex vivo removal/in vitro treatment elicited an efficient antitumor immune response if DX was added during the in vitro incubation, although cryostored cells were less efficient than freshly recovered tumor cells. The omission of DX curtailed the efficacy of the vaccine ( A). As an alternative to this ex vivo/in vitro protocol of vaccine generation, we injected DX into palpable (>125 mm 3 ) subcutaneous CT26 tumors esta..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "MATERIALS AND METHODS",
"text": "CT26, PROb, B16F10, B16/F10.9-OVA, and B16F10A2/gp100 (B16F10 transfected with gp100 and HLA A2.1 and selected in 50 µg/ml hygromicin and G418) cells were cultured at 37°C under 5% CO 2 in RPMI 1640 medium supplemented with 10% FCS, penicillin, streptomycin, 1 mM pyruvate, and 10 mM Hepes in the presence of DX for 24 h (25 µM for CT26, 30 µM for PROb, and 2.5 µM for B16F10 and its derivatives), 5 µM daunorubicin for 24 h (GE Healthcare), 1 µM idarubicin for 24 h (Aventis), 30 µM MC for 48 h (Sanofi-Synthelabo), and/or 100 µM zVAD-fmk for 24 h (Bachem). Necrosis was induced by one freeze-thaw cycle in liquid N 2 and a 37°C water bath. CT26 cells were stably transfected with vector only (Neo) or with a pcDNA3.1 vector encoding p35 (, ). In one experiment, 100 µM DX was added for 15 min to cells cultured in MC for 2 d. The incorpora..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Cell death assays.",
"text": "Cells were trypsinized and subjected to cytofluorometric analysis with a FACSVantage after staining with 2.5 µM DAPI for 10 min (Invitrogen) for determination of cell viability, and annexin V was conjugated with fluorescein isothiocyanate (Bender Medsystems) for the assessment of phosphatidylserine exposure (, ). TUNEL assays were performed on cells (let to adhere for 1 h in PBS on polylysine-coated slides [O. Kindler GmbH] and fixed with 4% paraformaldehyde for 30 min) using the In situ Cell Death Detection Kit (Roche) and a fluorescence microscope (IRE2; Leica) equipped with a camera (DC300F; Leica). For electron microscopy, cells (fixed for 1 h at 4°C in 2.5% glutaraldehyde in phosphate buffer, pH 7.4, washed, and fixed again in 2% osmium tetroxide and then embedded in Epon) and ultrathin sections (80 nm) were stained with uranyl acetate and lead citrate and examined wit..."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Immunoblot analyses.",
"text": "Cells were washed with cold PBS at 4°C and lysed in a buffer containing 50 mM Tris HCl, pH 6.8, 10% glycerol, and 2% SDS. Primary antibodies detecting activated caspase 3 (dilution 1/1,000; Cell Signaling Technology), HSP70 (dilution 1/1,000; Stratagene) or 2 µg/ml HMGB-1 (BD Biosciences) were revealed with the appropriate horseradish peroxidase-labeled secondary antibody (Southern Biotechnology Associates, Inc.) and detected by enhanced chemiluminescence (Pierce Chemical Co.). Anti-actin or anti-GAPDH (Chemicon) was used to control equal loading."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Antitumor vaccination and assessment of tumor growth in vivo.",
"text": "All animals were maintained in specific pathogen-free conditions, and all experiments followed the Federation of European Laboratory Animal Science Association guidelines. 3 × 10 6 treated CT26 cells were inoculated subcutaneously in 200 µl PBS into 6-wk-old female BALB/c mice (Charles River Laboratories) into the lower flank, whereas 5 × 10 5 untreated control cells were inoculated into the contralateral flank ( ). PROb cells (3 × 10 6 treated cells and 10 6 control cells) were injected subcutaneously into the lower flanks of syngeneic 2-mo-old BDIX rats (Charles River Laboratories), and B16A2 and B16F10 (1.8 × 10 5 treated cells and 3 × 10 4 control cells) were injected into 6-wk-old female HHD2 transgenic mice (bred in the Institut Gustave Roussy [IGR] animal facility) and C57BL/6 mice (Charles River Laboratories), respectively. For the tumorigenicity..."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Assessment of specific T cell responses.",
"text": "The CTL response against the immunodominant CT26-specific AH1 H2L d -restricted peptide (SPSYVYHQF; Neosystem; reference ) was determined. Splenocytes were isolated from immunized mice 10 d after injection of apoptotic cells and restimulated in vitro for 5 d with or without 5 µg/ml AH1 in the presence of syngeneic irradiated naive spleen cells. The cytotoxic activity was determined during a classic 5-h in vitro 51 Cr-release assay using 51 Cr-P815 tumor cells loaded with 50 µM AH1 peptide as target cells ( ). The percentage of specific lysis was calculated as 100 × (experimental release - spontaneous release)/(maximal release - spontaneous release). Maximum release was obtained by adding 10% Triton X-405 to target cells, and spontaneous release was determined by incubating target cells in medium alone. 5-6-wk-old female C57BL/6 mice (H-2 b ) were injec..."
}
],
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"@type": "HowToTool",
"name": "Immunogenicity of caspase-dependent, DX-induced cell death"
},
{
"@type": "HowToTool",
"name": "Immunogenicity of caspase-dependent, DX-induced cell death"
},
{
"@type": "HowToTool",
"name": "Critical role of DCs in the immune response elicited by dying tumor cells"
},
{
"@type": "HowToTool",
"name": "Critical role of DCs in the immune response elicited by dying tumor cells"
},
{
"@type": "HowToTool",
"name": "Critical role of DCs in the immune response elicited by dying tumor cells"
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{
"@type": "HowToTool",
"name": "DX-elicited apoptosis is immunogenic in several tumor models"
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"@type": "HowToTool",
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"@type": "HowToSupply",
"name": "Immunogenicity of caspase-dependent, DX-induced cell death"
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"@type": "HowToSupply",
"name": "Critical role of DCs in the immune response elicited by dying tumor cells"
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"@type": "HowToSupply",
"name": "Ex vivo and in vivo induction of immunogenic apoptosis"
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"headline": "Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death",
"datePublished": "2005",
"author": [
{
"@type": "Person",
"name": "Noelia Casares"
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{
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"name": "Marie O. Pequignot"
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{
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"name": "Antoine Tesniere"
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"name": "François Ghiringhelli"
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"name": "Stéphan Roux"
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"name": "Nathalie Chaput"
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"name": "Elise Schmitt"
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{
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"name": "Ahmed Hamai"
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{
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"name": "Sandra Hervas-Stubbs"
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"identifier": "10.1084/jem.20050915"
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